LIBRARY 

OF  THE 

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-B  ft  .  ^ 

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OCT18 
OCT 

JAN 


DECS 


Pentland's  Students   Manuals. 


MANUAL   OF    BACTERIOLOGY. 


NUNQUAM    ALIUD    NATURA,    ALIUD   SAPIENTIA    DIGIT. 


MANUAL 


OF 


BACTEBIOLOGY 


BY 

ROBERT  MUIR,  M.A.,  M.D.,  F.R. C.P.ED. 

PROFESSOR   OF   PATHOLOGY,    UNIVERSITY   OF  GLASGOW 


AND 


JAMES   RITCHIE,  M.A.,  M.D.,  B.Sc. 

SUPERINTENDENT    OF   THE    ROYAL    COLLEGE    OF   PHYSICIANS'    LABORATORY,    EDINBURGH 
FORMERLY   PROFESSOR   OF   PATHOLOGY   IN   THE   UNIVERSITY   OF  OXFORD 


FOURTH  EDITION 


WITH  ONE  HUNDRED  &  SEVENTY-ONE   ILLUSTRATIONS 


UNIVERSITY 

of 


NEW  YORK 

THE    MACMILLAN    COMPANY 

EDINBURGH   AND   LONDON:    YOUNG   J.  PENTLAND 
1907 


EDINBURGH  :     PRINTED    FOR   YOUNG   J.   PENTLAND,   II    TEVIOT    PLACE 

AND    21    WARWICK    LANE,    PATERNOSTER    ROW,    LONDON,    E.G., 

BY   R.    AND    R.    CLARK,    LIMITED 


A II  rights  reserved 


PREFACE  TO  THE  FOURTH  EDITION. 

IN  the  present  edition  the  whole  subject  has  been  carefully 
revised.  During  the  five  years  since  the  last  edition  was 
published,  valuable  additions  to  our  knowledge  have  been 
made  in  practically  every  department,  whilst  in  the  case  of 
several  diseases  there  have  been  discoveries  of  the  highest 
importance.  Our  object  has  been  to  incorporate  this  new 
matter  and  at  the  same  time  to  maintain  the  primary  object 
of  the  work  as  a  text-book  for  students  of  medicine.  Thus 
whilst  we  have  dealt  with  all  the  facts  having  a  direct  bearing 
on  clinical  medicine  we  have  also  given  considerable  prominence 
to  matters  at  present  under  discussion  from  the  scientific  point 
of  view.  In  this  way  we  have  endeavoured  to  give  a  faithful 
representation  of  the  subject  as  it  at  present  stands  both  in 
its  practical  and  theoretical  aspects.  In  the  case  of  several 
diseases  which  up  till  recent  times  have  been  investigated  by 
purely  bacteriological  methods  there  is  now  considerable  evidence 
that  the  causal  agent  is  of  protozoal  nature.  Amongst  such 
conditions  the  most  important  are  those  in  which  spirochsetes 
are  concerned,  syphilis  and  the  relapsing  fevers  being  outstand- 
ing examples.  As,  however,  the  exact  biological  relationships 
of  these  organisms  are  still  matters  of  dispute  we  have  kept 

185888 


vi  PREFACE   TO   THE   FOURTH   EDITION 

the  diseases  in  question  in  the  original  arrangement.  In  the 
appendix  will  be  found  an  additional  chapter  dealing  with 
trypanosomiasis  and  allied  affections.  A  number  of  new 
illustrations  have  been  added  throughout  the  book,  and  the 
bibliography  has  been  brought  up  to  date. 

October  1907. 


PEEFACE  TO  THE  FIRST  EDITION. 

THE  science  of  Bacteriology  has,  within  recent  years,  become 
so  extensive,  that  in  treating  the  subject  in  a  book  of  this  size 
we  are  necessarily  restricted  to  some  special  departments,  unless 
the  description  is  to  be  of  a  superficial  character.  Accordingly, 
as  this  work  is  intended  primarily  for  students  and  practitioners 
of  medicine,  only  those  bacteria  which  are  associated  with 
disease  in  the  human  subject  have  been  considered.  We  have 
made  it  a  chief  endeavour  to  render  the  work  of  practical  utility 
for  beginners,  and,  in  the  account  of  the  more  important 
methods,  have  given  elementary  details  which  our  experience  in 
the  practical  teaching  of  the  subject  has  shown  to  be  necessary. 

In  the  systematic  description  of  the  various  bacteria,  an 
attempt  has  been  made  to  bring  into  prominence  the  evidence  of 
their  having  an  etiological  relationship  to  the  corresponding 
diseases,  to  point  out  the  general  laws  governing  their  action  as 
producers  of  disease,  and  to  consider  the  effects  in  particular 
instances  of  various  modifying  circumstances.  Much  research 
on  certain  subjects  is  so  recent  that  conclusions  on  many  points 
must  necessarily  be  of  a  tentative  character.  We  have,  therefore, 
in  our  statement  of  results  aimed  at  drawing  a  distinction 
between  what  is  proved  and  what  is  only  probable. 

In  an  Appendix  we  have  treated  of  four  diseases ;  in  two  of 
these  the  causal  organism  is  not  a  bacterium,  whilst  in  the  other 
two  its  nature  is  not  yet  determined.  These  diseases  have  been 

vii 


viii  PREFACE   TO   THE   FIRST   EDITION 

included  on  account  of  their  own  importance  and  that  of  the 
pathological  processes  which  they  illustrate. 

Our  best  thanks  are  due  to  Professor  Greenfield  for  his  kind 
advice  in  connection  with  certain  parts  of  the  work.  We  have 
also  great  pleasure  in  acknowledging  our  indebtedness  to 
"Dr.  Patrick  Manson,  who  kindly  lent  us  the  negatives  or  pre- 
parations from  which  Figs.  160-165  have  been  executed. 

As  we  are  convinced  that  to  any  one  engaged  in  practical 
study,  photographs  and  photomicrographs  supply  the  most  useful 
and  exact  information,  we  have  used  these  almost  exclusively  in 
illustration  of  the  systematic  description.  These  have  been 
executed  in  the  Pathological  Laboratory  of  the  University  of 
Edinburgh  by  Mr.  Richard  Muir.  The  line  drawings  were 
prepared  for  us  by  Mr.  Alfred  Robinson,  of  the  University 
Museum,  Oxford. 

To  the  volume  is  appended  a  short  Bibliography,  which, 
while  having  no  pretension  to  completeness,  will,  we  hope,  be  of 
use  in  putting  those  who  desire  further  information  on  the  track 
of  the  principal  papers  which  have  been  published  on  each  of 
the  subjects  considered 

— 
June  1897. 


CONTENTS. 

CHAPTER    I. 
GENERAL  MORPHOLOGY  AND  BIOLOGY. 

PAGE 

INTRODUCTORY — Terminology — Structure  of  the  bacterial  cell — 
Reproduction  of  bacteria  —  Spore  formation  —  Motility  — 
Minuter  structure  of  the  bacterial  protoplasm  —  Chemical 
composition  of  bacteria  —  Classification  —  Food  supply — Re- 
lation of  bacteria  to  moisture,  gaseous  environment,  tempera- 
ture, and  light — Conditions  affecting  bacterial  motility  — 
Effects  of  bacteria  in  nature — Methods  of  bacterial  action — 
Variability  among  bacteria  .....  1 

CHAPTER    II. 
METHODS  OF  CULTIVATION  OP  BACTERIA. 

Introductory — Methods  of  sterilisation — Preparation  of  culture 
media — Use  of  the  culture  media — Methods  of  the  separation 
of  aerobic  organisms — Principles  of  the  culture  of  anaerobic 
organisms  —  Miscellaneous  methods  —  General  laboratory 
rules  ........  25 

CHAPTER    III. 

MICROSCOPIC  METHODS — GENERAL  BACTERIOLOGICAL 
DIAGNOSIS — INOCULATION  OF  ANIMALS. 

The  microscope — Examination  of  hanging-drop  cultures — Film  pre- 
parations— Examination  of  bacteria  in  tissues — The  cutting 
b  ix 


CONTENTS 

PAGE 

of  sections — Staining  principles — Mordants  and  decolorisers 
— Formulae  of  stains — Gram's  method  and  its  modifications 
— Stain  for  tubercle  and  other  acid-fast  bacilli — Staining  of 
spores  and  flagella — The  Romanowsky  stains — Observation  of 
agglutination  and  sedimentation — Method  of  measuring  the 
phagocytic  capacity  of  the  leucocytes — Routine  bacteriological 
examination — Methods  of  inoculation — Autopsies  on  animals  85 


CHAPTER    IV. 
BACTERIA  IN  AIR,  SOIL,  AND  WATER.     ANTISEPTICS. 

Air :  Methods  of  examination  —  Results.  Soil :  Methods  of 
Examination— Varieties  of  bacteria  in  soil.  Water  :  Methods 
of  examination — Bacteria  in  water — Bacterial  treatment  of 
sewage.  Antiseptics  :  Methods  of  investigation — The  action 
of  antiseptics — Certain  particular  antiseptics .  .  .126 

CHAPTER   V. 

RELATIONS  OF  BACTERIA  TO  DISEASE — THE  PRODUCTION 
OP  TOXINS  BY  BACTERIA. 

Introductory  —  Conditions  modifying  pathogenicity — Modes  of 
bacterial  action — Tissue  changes  produced  by  bacteria — Local 
lesions — General  lesions — Disturbance  of  metabolism  by 
bacterial  action — The  production  of  toxins  by  bacteria,  and 
the  nature  of  these — Allied  vegetable  and  animal  poisons — 
The  theory  of  toxic  action  .....  149 

CHAPTER   VI. 
INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS. 

The  relations  of  inflammation  and  suppuration — The  bacteria  of 
inflammation  and  suppuration — Experimental  inoculation — 
Lesions  in  the  human  subject — Mode  of  entrance  and  spread 
of  pyogenic  bacteria — Ulcerative  endocarditis — Acute  suppur- 
ative  periostitis — Erysipelas — Conjunctivitis — Acute  rheu- 
"matism — Vaccination  treatment  of  infections  by  the  pyogenic 
cocci — Methods  of  examination  in  inflammatory  and  suppur- 
ative  conditions  .  172 


CONTENTS  xi 

CHAPTER   VII. 

INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS,  CONTINUED  : 

THE  ACUTE  PNEUMONIAS,  EPIDEMIC  CEREBRO-SPINAL 

MENINGITIS. 

PAGE 

Introductory  —  Historical  —  Bacteria  in  pneumonia  —  Fraenkel's 
pneumococcus — Friedlaender's  pneumococcus — Distribution  of 
pneumobacteria  —  Experimental  inoculation  —  Pathology  of 
pneumococcus — Methods  of  examination.  Epidemic  cerebro- 
spinal  meningitis  ......  196 

CHAPTER   VIII. 

.GONORRHCEA,    SOFT    SORE,    SYPHILIS. 

The  gonococcus  —  Microscopical  characters  —  Cultivation  —  Rela- 
tions to  the  disease — Its  toxin — Distribution — Gonococcus  in 
joint  affections — Methods  of  diagnosis — Soft  sore — Syphilis — 
Spirochsete  pallida — Transmission  of  the  disease  to  animals  .  219 

CHAPTER    IX. 
TUBERCULOSIS. 

Historical — Tuberculosis  in  animals — Tubercle  bacillus — Staining 
reactions — Cultivation  of  tubercle  bacillus — Powers  of  resist- 
ance— Action  on  the  tissues — Histology  of  tuberculous  nodules 
— Distribution  of  bacilli — Bacilli  in  tuberculous  discharges — 
Experimental  inoculation — Varieties  of  tuberculosis — Other 
acid-fast  bacilli — Action  of  dead  tubercle  bacilli — Sources  of 
human  tuberculosis — Toxins  of  the  tubercle  bacillus — Koch's 
tuberculin — Active  immunisation  against  the  tubercle  bacillus 
— Koch's  Tuberculin-R — Agglutinative  phenomena — Methods 
of  examination  .  .  .  .  •.  .  .  235 

CHAPTER   X. 

LEPROSY. 

Pathological  changes — Bacillus  of  leprosy — Position  of  the  bacilli 

—Relations  to  the  disease— Methods  of  diagnosis        .  .       267 


xii  CONTENTS 

CHAPTER    XI. 
GLANDERS  AND  RHINOSCLEROMA. 

PAGE 

Glanders  :  The  natural  disease — The  glanders  bacillus — Cultiva- 
tion of  glanders  bacillus — Powers  of  resistance — Experimental 
inoculation — Action  on  the  tissues — Mode  of  spread — Mallein 
and  its  preparation — Methods  of  examination.  Rhinoscleroma  275 

CHAPTER    XII. 

ACTINOMYCOSIS    AND    ALLIED    DISEASES. 

Characters  of  the  actinomyces— Tissue  lesions— Distribution  of 
lesions — Cultivation  of  actinomyces — Varieties  of  actinomyces 
and  allied  forms  —  Experimental  inoculation  —  Methods  of 
examination  and  diagnosis — Madura  disease  .  .  .  286 

CHAPTER  -XIII. 
ANTHRAX. 

Historical  summary — Bacillus  anthracis — Appearances  of  cultures 
— Biology — Sporulation — Natural  anthrax  in  animals — Ex- 
perimental anthrax — Anthrax  in  man — Pathology — Toxins  of 
the  bacillus  anthracis — Mode  of  spread  in  nature — Immunisa- 
tion of  animals  against  anthrax — Methods  of  examination  .  300 

CHAPTER    XIV. 

TYPHOID  FEVER — BACILLI  ALLIED  TO  THE  TYPHOID 
BACILLUS. 

Bacillus  typhosus — Morphological  characters — Characters  of  cul- 
tures— Bacillus  coli  communis — Reactions  of  b.  typhosus  and 
b.  coli — Pathological  changes  in  typhoid  fever — Suppuration 
in  typhoid  fever — Pathogenic  effects  produced  in  animals — 
The  toxic  products  of  typhoid  bacillus  —  Immunisation  of 
animals  —  Relations  of  bacilli  to  the  disease  —  Paratyphoid 
bacillus— Bacillus  enteritidis  (Gaertner)— Psittacosis  bacillus 
— Serum  diagnosis — Vaccination  against  typhoid — Methods  of 
examination  —  Bacteria  in  dysentery  —  Bacillus  enteritidis 
sporogenes — Summer  diarrhoea  .  .  .  .319 


CONTENTS  xiii 

CHAPTER   XV. 
DIPHTHERIA. 

PAGE 

Historical — General  facts  —  Bacillus  diphtherise  —  Microscopical 
characters  —  Distribution  —  Cultivation  —  Inoculation  experi- 
ments— The  toxins  of  diphtheria — Variations  in  virulence  of 
bacilli — Bacilli  allied  to  the  diphtheria  bacillus— Summary  of 
pathogenic  action — Methods  of  diagnosis  .  .  .  352 

CHAPTER    XVI. 
TETANUS. 

Introductory — Historical  —  Bacillus  tetani — Isolation  of  bacillus 
tetani — Characters  of  cultures — Conditions  of  growth — Patho- 
genic effects — Experimental  inoculation — Tetanus  toxins — 
Antitetanic  serum  —  Methods  of  examination  —  Malignant 
oedema — Characters  of  bacillus — Experimental  inoculation — 
Methods  of  diagnosis — Bacillus  botulinus — Quarter -evil — . 
Bacillus  serogenes  capsulatus  .  .  .  .  .371 

CHAPTER    XVII. 
CHOLERA. 

Introductory — The  cholera  spirillum — Distribution  of  the  spirilla — 
Cultivation — Powers  of  resistance — Experimental  inoculation 
Toxins  of  cholera  spirillum — Inoculation  of  human  subject — 
Immunity  —Methods  of  diagnosis — General  summary — Other 
spirilla  resembling  the  cholera  organism  —  Metchnikoff's 
spirillum — Finkler  and  Prior's  spirillum — Deneke's  spirillum  399 

CHAPTER   XVIII. 

INFLUENZA,  PLAGUE,  RELAPSING  FEVER,  MALTA  FEVER, 
YELLOW  FEVER. 

Influenza  bacillus — Microscopical  characters — Cultivation — Dis- 
tribution— Experimental  inoculation — Methods  of  examina- 


xiv  CONTENTS 


PAGE 


tiori:  —  Bacillus  of  plague  —  Microscopical  characters  —  Cultiva- 
tion —  Anatomical  changes  produced  and  distribution  of 
bacilli  —  Experimental  inoculation  —  Paths  and  mode  of  in- 
fection —  Toxins,  immunity,  etc.  —  Methods  of  diagnosis  — 
Relapsing  fever  and  African  tick  fever  —  Characters  of  the 
spirillum  —  Relations  to  the  disease  —  Immunity  —  African 
tick  fever  —  Malta  fever  —  Micrococcus  melitensis  —  Relations  to 
the  disease  —  Mode  of  spread  of  the  disease  —  Methods  of 
diagnosis  —  Yellow  fever  —  Etiology  of  yellow  fever  .  .  420 


CHAPTER    XIX. 
IMMUNITY. 

Introductory  —  Acquired  immunity  —  Artificial  immunity- 
Varieties —  Active  immunity — Methods  of  production — At- 
tenuation and  exaltation  of  virulence — Passive  immunity — 
Action  of  the  serum — Antitoxic  serum — Standardising  of 
toxins  and  of  antisera — Nature  of  antitoxic  action — Ehrlich's 
theory  of  the  constitution  of  toxins — Antibacterial  serum — 
Bactericidal  and  lysogenic  action  —  Haemolytic  and  other 
sera  —  Methods  of  hsemolytic  tests  —  Opsonic  action  —  Ag- 
glutination— Precipitins — Therapeutic  effects  of  anti-sera — 
Theories  as  to  acquired  immunity — Ehrlich's  side-chain  theory 
—  Serum  anaphylaxis — Theory  of  phagocytosis  —  Natural 
immunity — Natural  bactericidal  powers — Natural  suscepti- 
bility to  toxins  .......  456 


APPENDIX   A. 
SMALLPOX  AND  VACCINATION. 

Jennerian  vaccination — Relationship  of  smallpox  to  cowpox — 
Micro-organisms  associated  with  smallpox — The  nature  of 
vaccination  .......  503 

APPENDIX    B. 
HYDROPHOBIA. 

Introductory — Pathology— The  virus  of  hydrophobia — Prophylaxis 

— Antirabic  serum — Methods   .  .  .510 


CONTENTS  xv 

APPENDIX   C. 
MALARIAL  FEVER. 

The  malarial  parasite— The  cycle  of  the  malarial  parasite  in  man 
— The  cycle  in  the  mosquito — Varieties  of  the  malarial  para- 
site— General  considerations — The  pathology  of  malaria — 
Methods  of  examination  .....  521 


APPENDIX    D. 
AMCEBIC  DYSENTERY. 

Amoebic  dysentery — Characters  of  the  amoeba — Distribution  of  the 

amoebae — Experimental  inoculation — Methods  of  examination        537 

APPENDIX    E. 
TRYPANOSOMIASIS — KALA-AZAR — PIROPLASMOSIS. 

The  pathogenic  trypanosomes — General  morphology  of  the  trypano- 
somata — Trypanosoma  Lewisi — Nagana  or  tse-tse  fly  disease 
— Trypanosoma  of  sleeping  sickness — Trypanosoma  gambiense 
— Kdla-dzar — Dehli  sore — Piroplasmosis  .  .  .  544 


BIBLIOGRAPHY    .......       571 

INDEX  593 


LIST  OF  ILLUSTRATIONS. 


FIG.  PAGE 

1.  Forms  of  bacteria  .            .             .             .             .             .13 

2.  Hot-air  steriliser  .            .  .          .            .             .             .27 

3.  Koch's  steam  steriliser  .  ....         27 

4.  Autoclave        x  .  .             .             .             .29 

5.  Steriliser  for  blood  serum  .             .            .             .             .30 

6.  Meat  press  .            .            ...            „            .             .         31 

7.  Hot-water  funnel  .            .    -                     .            .             .35 

8.  Blood  serum  inspissator  .             .             .             .            .         40 

9.  Potato  jar  .             .....             .             .45 

10.  Cylinder  of  potato  cut  obliquely  ..            .             .             .45 

11.  Ehrlich's  tube  containing  piece  of  potato  .             .          -  .         45 

12.  Apparatus  for  filling  tubes         .  .             .                                    48 

13.  Tubes  of  media  ......         48 

14.  Platinum  wires  in  glass  handles  .             .                          .49 

15.  Method  of  inoculating  solid  tubes         .  .             .             .50 

16.  Rack  for  platinum  needles         .....         50 

17.  Petri's  capsule    .......         51 

18.  Koch's  levelling  apparatus  for  use  in  preparing  plates  .         54 

19.  Koch's  levelling  apparatus          .....         54 

20.  Esmarch's  tube  for  roll  culture  .             .             .             .55 

21.  Apparatus  for  supplying  hydrogen  for  anaerobic  cultures        .         58 

22.  Esmarch's  roll-tube  adapted  for  culture  containing  anaerobes          59 

23.  Bulloch's  apparatus  for  anaerobic  plate  cultures  .             ._        59 

24.  Flask  for  anaerobes  in  liquid  media       .  .                      .     .         61 

25.  Flask  arranged  for  culture  of  anaerobes  which  develop  gas      .         62 

26.  .Tubes  for  anaerobic  cultures  on  the  surface  of  solid  media       .         62 

27.  Slides  for  hanging-drop  cultures  .             .             .             .63 

28.  Graham  Brown's  chamber  for  anaerobic  hanging-drops  .         64 

29.  Apparatus  for  counting  colonies  .             .             .             .65 

30.  Wright's  250  c.mm.  pipette  fitted  with  nipple  .         66 

31.  Geissler's  vacuum  pump  for  filtering  cultures  .  .             .70 

32.  Chamberland's  candle  and  flask  arranged  for  filtration  .         70 

xvii 


xviii  LIST   OF   ILLUSTRATIONS 

FIG.  PAGE 

33.  Chamberland's  bougie  with  lamp  funnel  .  .  .71 

34.  Bougie  inserted  through  rubber  stopper  .  .  .71 

35.  Muencke's  modification  of  Chamberland's  filter  .  .         72 

36.  Flask  fitted  with  porcelain  bougie  for  filtering  large  quantities 

of  fluid            .             .             .             .             .  .  .73 

37.  Flask  for  filtering  small  quantities  of  fluid       .  73 

38.  Tubes  for  demonstrating  gas-formation  by  bacteria  .  .         76 

39.  Geryk  air-pump  for  drying  in  vacuo      .            .  . '  .79 

40.  Reichert's  gas  regulator              .             .             .  .  .80 

41.  Hearson's  incubator  for  use  at  37°  C.     .             .  .  .         81 

42.  Cornet's  forceps  for  holding  cover-glasses          .  .  .87 

43.  Needle  with  square  of  paper  on  end  for  manipulating  paraffin 

sections  .......         92 

44.  Syphon  wash-bottle  for  distilled  water  .  .  .96 

45.  Wright's  5  c.mm.  pipette  .  .  .  .  .108 

46.  Tubes  used  in  testing  agglutinating  and  sedimenting  properties 

of  serum         .......       110 

47.  Wright's  blood-capsule  .  .  .  .  .114 

48.  Test-tube  and  pipette  arranged  for  obtaining  fluids  containing 

bacteria  .......       116 

49.  Hollow  needle  for  intraperitoneal  inoculations  .  .       121 

50.  Hesse's  tube       .  .....       127 

51.  Petri's  sand  filter  .  .  .  .  .  .128 

52.  Staphylococcus   pyogenes    aureus,    young    culture   on    agar. 

xlOOO            .......  175 

53.  Two  stab  cultures  of  staphylococcus  pyogenes  aureus  in  gelatin  175 

54.  Streptococcus  pyogenes,  young  culture  on  agar.      x  1000         .  176 

55.  Culture  of  the  streptococcus  pyogenes  on  an  agar  plate            .  177 

56.  Bacillus  pyocyaneus  ;  young  culture  on  agar.      x  1000             .  177 

57.  Micrococcus  tetragenus.      x  1000  .  .  .  .181 

58.  Streptococci  in  acute  suppuration,      x  1000      .             .             .  184 

59.  Minute  focus  of  commencing  suppuration  in  brain,      x  50       .  186 

60.  Secondary  infection  of  a  glomerulus  of  kidney  by  the  staphylo- 

coccus aureus.      x300  .  .  .  .  .187 

61.  Section  of  a  vegetation  in  ulcerative  endocarditis,      x  600      .       189 

62.  Film  preparation  from  a  case  of  acute  conjunctivitis,  showing 

the  Koch- Weeks  bacilli,      x  1000      .  .  .  .192 

63.  Film  preparation  of  conjunctival  secretion  showing  the  diplo- 

bacillus  of  conjunctivitis,      x  1000    .  .  .  .192 

64.  Film  preparation  of  pneumonic  sputum,  showing  numerous 

pneumococci  (Fraenkel's).      x  1000    .  .  .  .199 

65.  Friedlander's    pneumobacillus,    from    exudate  in    a  case   of 

pneumonia.      x  1000  ...  .       200 

66.  Fraenkel's  pneumococcus  in  serous  exudation.      x  1000  .       200 

67.  Stroke  culture  of  Fraenkel's  pneumococcus  on  blood  agar        .       201 


LIST   OF   ILLUSTRATIONS  xix 

HO.  PAGE 

68.  Fraenkel's  pneumococcus  from  a  pure  culture  on  blood  agar. 

xlOOO  ....  .       202 

69.  Stab  culture  of  Friedlander's  pneumobacillus  .         ,-   .       203 

70.  Friedlander's  pneumobacillus,  from  a  young  culture  on  agar. 

xlOOO  .......       203 

71.  Capsulated  pneumococci  in  blood  taken  from  the  heart  of  a 

rabbit,      x  1000        .  .  .  .  .  .206 

72.  Film  preparation  of  exudation  from  a  case  of  meningitis,    x  1000      213 

73.  Pure  culture  of  diplococcus  intracellularis      .  .  .       214 

74.  Portion  of  film  of  gonorrhceal  pus.      x  1000    .  .  .       220 

75.  Gonococci,  from  a  pure  culture  on  blood  agar.      x  1000          .       221 

76.  Film  preparations  of  pus  from  soft  chancre,  showing  Ducrey's 

bacillus.      x!500      .  .  .  .  .  .227 

77.  Ducrey's  bacillus,      x  1500      .  .  .  .  .228 
78  and  79.  Film  preparations  from  juice  of  hard  chancre  showing 

spirochsete  pallida.      xlOOO  ....       230 

80.  Section  of  spleen  from  a  case  of  congenital  syphilis,  showing 

spirochsete  pallida.      x  1000  .  .  .  .231 

81.  Spirochaete  refringens.      x  1000  .  .  .  .       231 

82.  Tubercle  bacilli,  from  a  pure  culture  on  glycerin  agar.      x  1000       237 

83.  Tubercle  bacilli  in  phthisical  sputum,      x  1000          .  .       238 

84.  Cultures  of  tubercle  bacilli  on  glycerin  agar   .  .  .       240 

85.  Tubercle  bacilli  in  section  of  human  lung  in  acute  phthisis. 

xlOOO  .......       244 

86.  Tubercle  bacilli  in  giant-cells.      x  1000  .  .       245 

87.  Tubercle  bacilli  in  urine,      x  1000       .  .  .  .246 

88.  Moeller's  Timothy-grass  bacillus,      x  1000     .  .  .253 

89.  Cultures  of  acid-fast  bacilli  grown  at  room  temperature         .       253 

90.  Smegma  bacilli.      x  1000          .  .  .  .  .254 

91.  Section  through  leprous  skin,  showing  the  masses  of  cellular 

granulation  tissue  in  the  cutis.      x  80  .  .       268 

92.  Superficial  part  of  leprous  skin,      x  500          .  .  .       270 

93.  High-power  view  of  portion  of  leprous  nodule  showing  the 

arrangement  of  the  bacilli  within  the  cells  of  the  granula- 
tion tissue.      xllOO              .             .             .  .  .271 

94.  Glanders  bacilli  amongst  broken-down  cells,  x  1000  .       277 

95.  Glanders  bacilli,      x  1000         .             .             .  .  .278 

96.  Actinomycosis  of  human  liver.      x  500            .  .  .       288 

97.  Actinomyces  in  human  kidney.      x  500           .  .  .       289 

98.  Colonies  of  actinomyces.      x  60            .             .  .  .       290 

99.  Cultures  of  the  actinomyces  on  glycerin  agar  .  .       293 

100.  Actinomyces,  from  a  culture  on  glycerin  agar.      x  1000         .       294 

101.  Shake  cultures  of  actinomyces  in  glucose  agar  .  .       295 

102.  Section  of  a  colony  of  actinomyces  from  a  culture  in  blood 

serum,      x  1500  295 


xx  LIST   OF   ILLUSTRATIONS 

FIG.  PAGE 

103.  Streptothrix  Madura.      x  1000  .  .  .  .298 

104.  Surface   colony   of  the  anthrax  bacillus  on  an  agar  plate. 

x30               .             .             .             .             .             .             .  302 

105.  Anthrax  bacilli,    arranged   in   chains,   from   a   twenty-four 

hours'  culture  on  agar  at  37°  C.      x  1000    .             .             .  303 

106.  Stab  culture  of  the  anthrax  bacillus  in  peptone-gelatin          .  303 

107.  Anthrax  bacilli  containing  spores,      x  1000    ,             .             .  305 

108.  Scraping  from  spleen  of  guinea-pig  dead  of  anthrax.      x  1000  307 

109.  Portion  of  kidney  of  a  guinea-pig  dead  of  anthrax.      x  300  .  309 

110.  A  large  clump  of  typhoid  bacilli  in  a  spleen.      x  500              .  320 

111.  Typhoid  bacilli,  from  a  young  culture  on  agar,  showing  some 

filamentous  forms.      x  1000              ....  321 

112.  Typhoid  bacilli,   from  a  young  culture  on  agar,   showing 

flagella.      x  1000       ......  322 

113.  Culture  of  the  typhoid  bacillus  and  of  the  bacillus  coli          .  323 

114.  Colonies  of  the  typhoid  bacillus  in  a  gelatin  plate.      x  15     .  324 

115.  Bacillus  coli  communis.      x  1000         .  325 

116.  Film    preparation    from    diphtheria    membrane  ;    showing 

numerous  diphtheria  bacilli.      x  1000         .             .             .  354 

117.  Section  through  a  diphtheritic  membrane  in  trachea,  show- 

ing diphtheria  bacilli,      x  1000       ....  355 

118.  Cultures  of  the  diphtheria  bacillus  on  an  agar  plate               .  357 

119.  Diphtheria   bacilli    from   a   twenty-four    hours'   culture  on 

agar.      x  1000  .  .  .  .  .357 

120.  Diphtheria  bacilli,  from  a  three  days'  agar  culture.      x  1000  358 

121.  Involution  forms  of  the  diphtheria  bacillus.      x  1000             .  358 

122.  Pseudo-diphtheria  bacillus  (Hofmann's).      x  1000      .             .  366 

123.  Xerosis  bacillus  from  a  young  agar  culture,      x  1000              .  367 

124.  Film   preparation   of  discharge   from    wound   in  a   case  of 

tetanus,  showing  several  tetanus  bacilli  of  "drumstick  " 

form.      x  1000           ......  373 

125.  Tetanus  bacilli,  showing  flagella.      x  1000     .             .             .  374 

126.  Spiral  composed  of  numerous  twisted  flagella  of  the  tetanus 

bacillus.      xlOOO     ....  .375 

127.  Tetanus  bacilli,  some  of  which  possess  spores.      x  1000          .  375 

128.  Stab  culture  of  the  tetanus  bacillus  in  glucose  gelatin            .  376 

129.  Film    preparation   from   the   affected    tissues    in   a   case   of 

malignant  oedema.      x  1000              ....  389 

130.  Bacillus  of  malignant  cedema,  showing  spores.      x  1000         .  390 

131.  Stab  cultures  in  agar — tetanus  bacillus,  bacillus  of  malignant 

redema,  and  bacillus  of  quarter-evil             .             .             .  391 

132.  Bacillus  of  quarter-evil,  showing  spores.      x  1000      .             .  397 

133.  Bacillus  serogenes  capsulatus               ....  398 

134.  Cholera  spirilla,  from  a  culture  on  agar  of  twenty- four  hours' 

growth.      x  1000      ......  400 


LIST   OF   ILLUSTRATIONS  xxi 

FIG.  PAGE 

135.  Cholera    spirilla    stained    to   show    the    terminal    flagella. 

xlOOO           .......  401 

136.  Cholera  spirilla  from  an  old  agar  culture.      x  1000    .             .  401 

137.  Puncture  culture  of  the  cholera  spirillum       .             .             .  403 

138.  Colonies  of  the  cholera  spirillum  on  a  gelatin  plate    .             .  404 

139.  MetchnikofFs  spirillum,      x  1000        .  .417 

140.  Puncture  cultures  in  peptone-gelatin                .             .             .  418 

141.  Finkler  and  Prior's  spirillum,      x  1000            .             .             .  419 

142.  Influenza  bacilli  from  a  culture  on  blood  agar.      x  1000         .  420 

143.  Film  preparation  from  a  plague  bubo.      x  1000           .             .  426 

144.  Bacillus  of  plague  from  a  young  culture  on  agar.        x  1000    .  427 

145.  Bacillus  of  plague  in  chains.      x  1000              .             .             .  427 

146.  Culture  of  the  bacillus  of  plague  on  4  per  cent  salt  agar. 

xlOOO           .......  428 

147.  Section  of  a  human  lymphatic  gland  in  plague.      x  50           .  430 

148.  Film  preparation  of  spleen  of  rat  after  inoculation  with  the 

bacillus  of  plague.      x  1000              ....  432 

149.  Spirilla  of  relapsing  fever  in  human  blood,      x  about  1000  .  439 

150.  Spirillum  Obermeieri  in  blood  of  infected  mouse.      x  1000    .  441 

151.  Film  of  human  blood  containing  spirillum  of  tick  fever. 

xlOOO           .......  444 

152.  Spirillum  of  human  tick  fever  (Spirillum  Duttoni;  in  blood 

of  infected  mouse,      x  1000              ....  445 

153.  Micrococcus  melitensis.      x  1000          ....  448 
154-159.  Various  phases  of  the  benign  tertian  parasite       .             .  525 
160-165.  Exemplifying  phases  of  the  malignant  parasite  .             .  526 

166.  Amoebae  of  dysentery    ..'....  538 

167.  Section  of  wall  of  liver  abscess,  showing  an  amceba  of  spherical 

form  with  vacuolated  protoplasm.      x  1000             .             .  54Q 

168.  Trypanosoma  Brucei  from  blood  of  infected  rat.     N"ote  in  two 

of  the  organisms  commencing  division  of  micronucleus  and 

undulating  membrane,      x  1000       ....  554 

169.  Trypanosoma.  gambiense  from  blood  of  guinea-pig.      x  1000  .  557 

170.  Leishman-Donovan  bodies  from  spleen  smear,      x  1000          .  564 

171.  Leishman-Donovan  bodies  within  endothelial  cell  in  spleen. 

x 1000  565 


MANUAL   OF   BACTERIOLOGY 


MANUAL  OF  BACTERIOLOGY, 


CHAPTER   I. 
GENERAL   MORPHOLOGY   AND   BIOLOGY. 

Introductory. — At  the  bottom  of  the  scale  of  living  things  there 
exists  a  group  of  organisms  to  which  the  name  of  bacteria  is 
usually  applied.  These  are  apparently  of  very  simple  structure 
and  may  be  subdivided  into  two  sub-groups,  a  lower  and  simpler 
and  a  higher  and  better  developed. 

The  lower  forms  are  the  more  numerous,  and  consist  of 
minute  unicellular  masses  of  protoplasm  devoid  of  chlorophyll, 
which  multiply  by  simple  fission.  Some  are  motile,  others  non- 
motile.  Their  minuteness  may  be  judged  of  by  the  fact  that  in 
one  direction  at  least  they  usually  do  not  measure  more  than 
1  p  (0-5 wo  incn)-  These  forms  can  be  classified  according  to 
their  shapes  into  three  main  groups — (1)  A  group  in  which  the 
shape  is  globular.  The  members  of  this  are  called  cocci.  (2)  A 
group  in  which  the  shape  is  that  of  a  straight  rod — the  pro- 
portion of  the  length  to  the  breadth  of  the  rod  varying  greatly 
among  the  different  members.  These  are  called  bacilli.  (3)  A 
group  in  which  the  shape  is  that  of  a  curved  or  spiral  rod. 
These  are  called  spirilla.  The  full  description  of  the  characters 
of  these  groups  will  be  more  conveniently  taken  later  (p.  11). 
In  some  cases,  especially  among  the  bacilli,  there  may  occur 
under  certain  circumstances  changes  in  the  protoplasm  whereby 
a  resting  stage  or  spore  is  formed. 

The  higher  forms  show  advance  on  the  lower  along  two  lines. 
(1)  On  the  one  hand  they  consist  of  filaments  made  up  of 
simple  elements  such  as  occur  in  the  lower  forms.  These 
1 


2         GENERAL   MORPHOLOGY   AND   BIOLOGY 

filaments  may  be  more  or  less  septate,  may  be  provided  with  a 
sheath,  and  may  show  branching  either  true  or  false.  The 
minute  structure  of  the  elements  comprising  these  filaments  is 
analogous  to  that  of  the  lower  forms.  Their  size,  however,  is 
often  somewhat  greater.  The  lower  forms  sometimes  occur  in 
filaments,  but  here  every  member  of  the  filament  is  independent, 
while  in  the  higher  forms  there  seems  to  be  a  certain  inter- 
dependence among  the  individual  elements.  For  instance, 
growth  may  occur  only  at  one  end  of  a  filament,  the  other 
forming  an  attachment  to  some  fixed  object.  (2)  The  higher 
forms,  moreover,  present  this  further  development  that  in  certain 
cases  some  of  the  elements  may  be  set  apart  for  the  reproduction 
of  new  individuals. 

Terminology. — The  term  bacterium  of  course  in  strictness 
only  refers  to  the  rod-shaped  varieties  of  the  group,  but  as  it 
has  given  the  name  bacteriology  to  the  science  which  deals  with 
the  whole  group,  it  is  convenient  to  apply  it  to  all  the  members 
of  the  latter,  and  to  reserve  the  term  bacillus  for  the  rod-shaped 
varieties.  Other  general  words,  such  as  germ,  microbe,  micro- 
organism, are  often  used  as  synonymous  with  bacterium,  though, 
strictly,  they  include  the  smallest  organisms  of  the  animal 
kingdom. 

While  no  living  organisms  lower  than  the  bacteria  are  known_ 
(though  the  occurrence  of  such  is  now  suspected),  the  upper 
limits  of  the  group  are  difficult  to  define,  and  it  is  further 
impossible  in  the  present  state  of  our  knowledge  to  give  other 
than  a  provisional  classification  of  the  forms  which  all  recognise 
to  be  bacteria.  The  division  into  lower  and  higher  forms, 
however,  is  fairly  well  marked,  and  we  shall  therefore  refer  to 
the  former  as  the  lower  bacteria,  and  to  the  latter  as  the  higher 
bacteria. 

Morphological  Relations. — The  relations  of  the  bacteria  to  the  animal 
kingdom  on  the  one  hand  and  to  the  vegetable  on  the  other  constitute  a 
somewhat  difficult  question.  It  is  best  to  think  of  there  being  a  group 
of  small,  unicellular  organisms,  which  may  represent  the  most  primitive 
forms  of  life  before  differentiation  into  animal  and  vegetable  types  had 
occurred.  This  would  include  the  flagellata  and  infusoria,  the  myxomy- 
cetes,  the  lower  algae,  and  the  bacteria.  To  the  lower  algae  the  bacteria 
possess  many  similarities.  These  algae  are  unicellular  masses  of  proto- 
plasm, having  generally  the  same  shapes  as  the  bacteria,  and  largely 
multiply  by  fission.  Endogenous  sporulation,  however,  does  not  occur, 
nor  is  motility  associated  with  the  possession  of  flagella.  Also  their 
protoplasm  differs  from  that  of  the  bacteria  in  containing  chlorophyll  and 
another  blue-green  pigment  called  phycocyan.  From  the  morphological 
resemblances,  however,  between  these  algae  and  the  bacteria,  and  from 
the  fact  that  fission  plays  a  predominant  part  in  the  multiplication  ojf 


STRUCTURE   OF   THE   BACTERIAL   CELL          3 

both,  they  have  been  grouped  together  in  one  class  as  the  Schizophyta 
or  splitting  plants  (German,  Spaltpflanzen).  And  of  the  two  divisions 
forming  these  Schizophyta  the  splitting  algee  are  denominated  the 
schizophycese  (German,  Spaltalgen),  while  the  bacteria  or  splitting  fungi 
are  called  the  schizomycetes  (German,  Spaltpilzen).  The  bacteria  are, 
therefore,  often  spoken  of  as  the  schizomycetes.  Certain  bacteria  which 
have  been  described  as  containing  chlorophyll  ought  probably  to  be 
grouped  among  the  schizophycesb. 


GENERAL  MORPHOLOGY  OF  THE  BACTERIA. 

The  Structure  of  the  Bacterial  Cell. — On  account  of  the 
minuteness  of  bacteria  the  investigation  of  their  structure  is 
attended  with  great  difficulty.  When  examined  under  the 
microscope,  in  their  natural  condition,  e.g.  in  water,  they  appear 
merely  as  colourless  refractile  bodies  of  the  different  shapes 
named.  Spore  formation  and  motility,  when  these  exist,  can 
also  be  observed,  but  little  else  can  be  made  out.  For  their 
proper  investigation  advantage  is  always  taken  of  the  fact  of 
their  affinities  for  various  dyes,  especially  those  which  are  usually 
chosen  as  good  stains  for  the  nuclei  of  animal  cells.  Certain 
points  have  thus  been  determined.  The  bacterial  cell  consists 
of  a  sharply  contoured  mass  of  protoplasm  which  reacts  to, 
especially  basic,  aniline  dyes  like  the  nucleus  of  an  animal  cell 
— though  from  this  fact  we  cannot  deduce  that  the  two  are 
identical  in  composition.  A  healthy  bacterium  when  thus 
stained  presents  the  appearance  of  a  finely  granular  or  almost 
homogeneous  structure.  The  protoplasm  is  surrounded  by  an 
envelope  which  can  in  some  cases  be  demonstrated  by  over- 
staining  a  specimen  with  a  strong  aniline  dye,  when  it  will  appear 
as  a  halo  round  the  bacterium.  This  envelope  may  sometimes 
be  seen  to  be  of  considerable  thickness.  Its  innermost  layer  is 
probably  of  a  denser  consistence,  and  sharply  contours  the 
contained  protoplasm,  giving  the  latter  the  appearance  of  being 
surrounded  by  a  membrane.  It  is  only,  however,  in  some  of 
the  higher  forms  that  a  true  membrane  occurs.  Sometimes  the 
outer  margin  of  the  envelope  is  sharply  defined,  in  which  case 
the  bacterium  appears  to  have  a  distinct  capsule,  and  is  known 
as  a  capsulated  bacterium  (vide  Fig.  1,  No.  3 ;  and  Fig.  64). 
The  cohesion  of  bacteria  into  masses  depends  largely  on  the 
character  of  the  envelope.  If  the  latter  is  glutinous,  then  a 
large  mass  of  the  same  species  may  occur,  formed  of  individual 
bacteria  embedded  in  what  appears  to  be  a  mass  of  jelly.  When 
this  occurs,  it  is  known  as  a  zooglcea  mass.  On  the  other  hand, 
if  the  envelope  has  not  this  cohesive  property  the  separation  of 


4         GENERAL   MORPHOLOGY   AND   BIOLOGY 

individuals  may  easily  take  place,  especially  in  a  fluid  medium 
in  which  they  may  float  entirely  free  from  one  another.  Many 
of  the  higher  bacteria  possess  a  sheath  which  has  a  much  more 
definite  structure  than  is  found  among  the  lower  forms.  It 
resists  external  influences,  possesses  elasticity,  and  serves  to  bind 
the  elements  of  the  organism  together. 

Reproduction  among  the  Lower  Bacteria. — When  a  bacterial 
cell  is  placed  in  favourable  surroundings  it  multiplies ;  as  has 
been  said,  this,  in  the  great  majority  of  cases,  takes  place  by 
simple  fission.  In  the  process  a  constriction  appears  in  the 
middle  and  a  transverse  unstained  line  develops  across  the 
protoplasm  at  that  point.  The  process  goes  on  till  two 
individuals  can  be  recognised,  which  may  remain  for  a  time 
attached  to  one  another,  or  become  separate,  according  to  the 
character  of  the  envelope,  as  already  explained.  In  most 
bacteria  growth  and  multiplication  go  on  with  great  rapidity. 
A  bacterium  may  reach  maturity  and  divide  in  from  twenty 
minutes  to  half  an  hour.  If  division  takes  place  only  every 
hour,  from  one  individual  after  twenty-four  hours  17,000,000 
similar  individuals  will  be  produced.  As  shown  by  the  results 
of  artificial  cultivation,  others,  such  as  the  tubercle  bacillus, 
multiply  much  more  slowly.  Sometimes  division  proceeds  so 
rapidly  that  the  young  individuals  do  not  reach  the  adult  size 
before  multiplication  again  occurs.  This  may  give  rise  to 
anomalous  appearances.  When  bacteria  are  placed  in  unfavour- 
able conditions  as  regards  food,  etc.,  growth  and  multiplication 
take  place  with  difficulty.  In  the  great  majority  of  cases  this  is 
evidenced  by  changes  in  the  appearance  of  the  protoplasm. 
Instead  of  it's  maintaining  the  regularity  of  shape  seen  in  healthy 
bacteria,  various  aberrant  appearances  are  presented.  This  occurs 
especially  in  the  rod -shaped  varieties,  where  flask- shaped  or 
dumb-bell-shaped  individuals  may  be  seen.  The  regularity  in 
structure  and  size  is  quite  lost.  The  appearance  of  the  protoplasm 
also  is  often  altered.  Instead  of,  as  formerly,  staining  well,  it 
does  not  stain  readily,  and  may  have  a  uniformly  pale,  homo- 
geneous appearance,  while  in  an  old  culture  only  a  small 
proportion  of  the  bacteria  may  stain  at  all.  Sometimes,  on  the 
other  hand,  a  degenerated  bacterium  contains  intensely  stained 
granules  or  globules  which  may  be  of  large  size.  Such  aberrant 
and  degenerate  appearances  are  referred  to  as  involution  forms. 
That  these  forms  really  betoken  degenerative  changes  is  shown 
by  the  fact  that,  on  their  being  again  transferred  to  favourable 
conditions,  only  slight  growth  at  first  takes  place.  Many 
individuals  have  undoubtedly  died,  and  the  remainder  which 


SPOKE   FORMATION  5 

live  and  develop  into  typical  forms  may  sometimes  have  lost 
some  of  their  properties. 

Reproduction  among  the  Higher  Bacteria.— Most  of  the  higher  bacteria 
consist  of  thread-like  structures  more  or  less  septate  and  often  surrounded 
by  a  sheath.  The  organism  is  frequently  attached  at  one  end  to  some 
object  or  to  another  individual.  It  grows  to  a  certain  length  and  then 
at  the  free  end  certain  cells  called  gonidia  are  cast  off  from  which  new 
individuals  are  formed.  These  gonidia  may  be  formed  by  a  division 
taking  place  in  the  terminal  element  of  the  filament  such  as  has  occurred 
in  the  growth  of  the  latter.  In  some  cases,  however,  division  takes 
place  in  three  dimensions  of  space.  The  gonidia  have  a  free  existence 
for  a  certain  time  before  becoming  attached,  and  in  this  stage  are 
sometimes  motile.  They  are  usually  rod-like  in  shape,  sometimes 
pyriform.  They  do  not  possess  any  special  powers  of  resistance. 

Spore  Formation. — In  certain  species  of  the  lower  bacteria, 
under  certain  circumstances,  changes  take  place  in  the  protoplasm 
which  result  in  the  formation  of  bodies  called  spores,  to  which 
the  vital  activities  of  the  original  bacteria  are  transferred. 
Spore  formation  occurs  chiefly  among  the  bacilli  and  in  some 
spirilla.  Its  commencement  in  a  bacterium  is  indicated  by  the 
appearance  in  the  protoplasm  of  a  minute  highly  refractile 
granule  unstained  by  the  ordinary  methods.  This  increases  in 
size,  and  assumes  a  round,  oval,  or  short  rod-shaped  form,  always 
shorter  but  often  broader  than  the  original  bacterium.  In  the 
process  of  spore  formation  the  rest  of  the  bacterial  protoplasm 
may  remain  unchanged  in  appearance  and  staining  power  for  a 
considerable  time  (e.g.  b.  tetani),  or,  on  the  other  hand,  it  may 
soon  lose  its  power  of  staining  and  ultimately  disappear,  leaving 
the  spore  in  the  remains  of  the  envelope  (e.g.  b.  anthracis). 
This  method  of  spore  formation  is  called  endogenous.  Bacterial 
spores  are  always  non-motile.  The  spore  may  appear  in  the 
centre  of  the  bacterium,  or  it  may  be  at  one  extremity,  or  a 
short  distance  from  one  extremity  (Fig.  1,  No.  11).  In  structure 
the  spore  consists  of  a  mass  of  protoplasm  surrounded  by  a  dense 
membrane.  This  can  be  demonstrated  by  methods  which  will 
be  described,  the  underlying  principle  of  which  is  the  prolonged 
application  of  a  powerful  stain.  The  membrane  is  supposed  to 
confer  on  the  spore  its  characteristic  feature,  namely,  great 
capacity  of  resistance  to  external  influences  such  as  heat  or 
noxious  chemicals.  Koch,  for  instance,  in  one  series  of  experi- 
ments, found  that  while  the  bacillus  anthracis  in  the  unspored 
form  was  killed  by  a  two  minutes'  exposure  to  1  per  cent  carbolic 
acid,  spores  of  the  same  organism  resisted  an  exposure  of  from 
one  to  fifteen  days. 

When  a  spore  is  placed  in  suitable  surroundings  for  growth 


6         GENEEAL   MORPHOLOGY  AND   BIOLOGY 

it  again  assumes  the  original  bacillary  or  spiral  form.  The 
capsule  dehisces  either  longitudinally,  or  terminally,  or  trans- 
versely. In  the  last  case  the  dehiscence  may  be  partial,  and  the 
new  individual  may  remain  for  a  time  attached  by  its  ends  to 
the  hinged  spore-case,  or  the  dehiscence  may  be  complete  and 
the  bacillus  grow  with  a  cap  at  each  end  consisting  of  half  the 
spore-case.  Sometimes  the  spore-case  does  not  dehisce,  but  is 
simply  absorbed  by  the  developing  bacterium. 

It  is  important  to  note  that  in  the  bacteria  spore  formation 
is  rarely,  if  ever,  to  be  considered  as  a  method  of  multiplication. 
In  at  least  the  great  majority  of  cases  only  one  spore  is  formed 
from  one  bacterium,  and  only  one  bacterium  in  the  first  instance 
from  one  spore.  Sporulation  is  to  be  looked  upon  as  a  resting 
stage  of  a  bacterium,  and  is  to  be  contrasted  with  the  stage 
when  active  multiplication  takes  place.  The  latter  is  usually 
referred  to  as  the  vegetative  stage  of  the  bacterium.  Regarding 
the  signification  of  spore  formation  in  bacteria  there  has  been 
some  difference  of  opinion.  According  to  one  view  it  may  be 
regarded  as  representing  the  highest  stage  in  the  vital  activity 
of  a  bacterium.  There  is  thus  an  alternation  between  the 
vegetative  and  spore  stage,  the  occurrence  of  the  latter  being 
necessary  to  the  maintenance  of  the  species  in  its  greatest 
vitality.  Such  a  rejuvenescence,  as  it  were,  through  sporulation, 
is  known  in  many  algae.  In  support  of  this  view  there  are 
certain  facts.  In  many  cases,  for  instance,  spore  formation  only 
occurs  at  temperatures  specially  favourable  for  growth  and 
multiplication.  There  is  often  a  temperature  below  which, 
while  vegetative  growth  still  takes  place,  sporulation  will  not 
occur  ;  and  in  the  case  of  b.  anthracis,  if  the  organism  be  kept 
at  a  temperature  above  the  limit  at  which  it  grows  best,  not 
only  are  no  spores  formed,  but  the  species  may  lose  the  power 
of  sporulation.  Furthermore,  in  the  case  of  bacteria  preferring 
the  presence  of  oxygen  for  their  growth,  an  abundant  supply  of 
this  gas  may  favour  sporulation.  It  is  probable  that  even  among 
bacteria  preferring  the  absence  of  oxygen  for  vegetative  growth, 
the  presence  of  this  gas  favours  sporulation.  Most  bacteriologists 
are,  however,  of  opinion  that  when  a  bacterium  forms  a  spore, 
it  only  does  so  when  its  surroundings,  especially  its  food  supply, 
become  unfavourable  for  vegetative  growth  ;  it  then  remains  in 
this  condition  until  it  is  placed  in  more  suitable  surroundings. 
Such  an  occurrence  would  be  analogous  to  what  takes  place 
under  similar  conditions  in  many  of  the  protozoa.  Often 
sporulation  can  be  prevented  from  taking  place  for  an  indefinite 
time  if  a  bacterium  is  constantly  supplied  with  fresh  food  (the 


SPOKE   FORMATION  7 

other  conditions  of  life  being  equal).  The  presence  of  substances 
excreted  by  the  bacteria  themselves  plays,  however,  a  more 
important  part  in  making  the  surroundings  unfavourable  than 
the  mere  exhaustion  of  the  food  supply.  A  living  spore  will 
always  develop  into  a  vegetative  form  if  placed  in  a  fresh  food 
supply.  With  regard  to  the  rapid  formation  of  spores  when 
the  conditions  are  favourable  for  vegetative  growth,  it  must  be 
borne  in  mind  that  in  such  circumstances  the  conditions  may 
really  very  quickly  become  unfavourable  for  a  continuance  of 
growth,  since  not  only  will  the  food  supply  around  the  growing 
bacteria  be  rapidly  exhausted,  but  the  excretion  of  effete  and 
inimical  matters  will  be  all  the  more  rapid. 

We  must  note  that  the  usually  applied  tests  of  a  body 
developed  within  a  bacterium  being  a  spore  are  (1)  its  staining 
reaction,  namely,  resistance  to  ordinary  staining  fluids,  but 
capacity  of  being  stained  by  the  special  methods  devised  for 
the  purpose  (vide  p.  102) ;  (2)  the  fact  that  the  bacterium 
containing  the  spore  has  higher  powers  of  resistance  against 
inimical  conditions  than  a  vegetative  form.  It  is  important  to 
bear  these  tests  in  mind,  as  in  some  of  the  smaller  bacteria 
especially,  it  is  very  difficult  to  say  whether  they  spore  or  not. 
There  may  appear  in  such  organisms  small  unstained  spots  the 
significance  of  which  it  is  very  difficult  to  determine. 

The  Question  of  Arthrosporo.us  Bacteria.— It  is  stated  by  Hueppe  that 
among  certain  organisms,  e.g.  some  streptococci,  certain  individuals  may, 
without  endogenous  sporulation,  take  on  a  resting  stage.  These  become 
swollen,  stain  well  with  ordinary  stains,  and  they  are  stated  to  have 
higher  power  of  resistance  than  the  other  forms  ;  further,  when  vegetative 
life  again  occurs  it  is  from  them  that  multiplication  is  said  to  take  place. 
From  the  fact  that  there  is  no  new  formation  within  the  protoplasm, 
but  that  it  is  the  whole  of  the  latter  which  participates  in  the  change, 
these  individuals  have  been  called  arthrospores.  The  existence  of  such 
special  individuals  amongst  the  lower  bacteria  is  extremely  problematical. 
They  have  no  distinct  capsule,  and  they  present  no  special  staining 
reactions,  nor  any  microscopic  features  by  which  they  can  be  certainly 
recognised,  while  their  alleged  increased  powers  of  resistance  are  very 
doubtful.  All  the  phenomena  noted  can  be  explained  by  the  undoubted 
fact  that  in  an  ordinary  growth  there  is  very  great  variation  among  the 
individual  organisms  in  their  powers  of  resistance  to  external  conditions. 

Motility. — As  has  been  stated,  many  bacteria  are  motile. 
Motility  can  be  studied  by  means  of  hanging-drop  preparations 
(vide  p.  63).  The  movements  are  of  a  darting,  rolling,  or 
vibratile  character.  The  degree  of  motility  depends  on  the 
species,  the  temperature,  the  age  of  the  growth,  and  on  the 
medium  in  which  the  bacteria  are  growing.  Sometimes  the 
movements  are  most  active  just  after  the  cell  has  multiplied, 


8         GENERAL   MORPHOLOGY   AND   BIOLOGY 

sometimes  it  goes  on  all  through  the  life  of  the  bacterium, 
sometimes  it  ceases  when  sporulation  is  about  to  occur.  Motility 
is  associated  with  the  possession  of  fine  wavy  thread-like 
appendages  called  flagella,  which  for  their  demonstration  require 
the  application  of  special  staining  methods  (vide  Fig.  1,  No.  12 ; 
and  Fig.  112).  They  have  been  shown  to  occur  in  many  bacilli 
and  spirilla,  but  only  in  a  few  species  of  cocci.  They  vary  in 
length,  but  may  be  several  times  the  length  of  the  bacterium, 
and  may  be  at  one  or  both  extremities  or  all  round.  When 
terminal  they  may  occur  singly  or  there  may  be  several.  The 
nature  of  these  flagella  has  been  much  disputed.  Some  have 
held  that,  unlike  what  occurs  in  many  alga?,  they  are  not  actual 
prolongations  of  the  bacterial  protoplasm,  but  merely  appendages 
of  the  envelope,  and  have  doubted  whether  they  are  really  organs 
of  locomotion.  There  is  now,  however,  little  doubt  that  they 
belong  to  the  protoplasm.  By  appropriate  means  the  central 
parts  of  the  latter  can  be  made  to  shrink  away  from  the  peripheral 
(vide  infra,  "  plasmolysis  ").  In  such  a  case  movement  goes  on 
as  before,  and  in  stained  preparations  the  flagella  can  be  seen 
to  be  attached  to  the  peripheral  zone.  It  is  to  be  noted  that 
flagella  have  never  been  demonstrated  in  non- motile  bacteria, 
while,  on  the  other  hand,  they  have  been  observed  in  nearly  all 
motile  forms.  There  is  little  doubt,  however,  that  all  cases  of 
motility  among  the  bacteria  are  not  dependent  on  the  possession 
of  flagella,  for  in  some  of  the  special  spiral  forms,  and  in  most 
of  the  higher  bacteria,  motility  is  probably  due  to  contractility 
of  the  protoplasm  itself. 

The  Minuter  Structure  of  the  Bacterial  Protoplasm.— Many  attempts 
have  been  made  to  obtain  deeper  information  as  to  the  structure  of  the 
bacterial  cell,  and  especially  as  to  its  behaviour  in  division.  These 
have  largely  turned  on  the  interpretation  to  be  put  on  certain  appear- 
ances which  have  been  observed.  These  appearances  are  of  two  kinds. 
First,  under  certain  circumstances  irregular  deeply-stained  granules  are 
observed  in  the  protoplasm,  often,  when  they  occur  in  a  bacillus,  giving 
the  latter  the  appearance  of  a  short  chain  of  cocci.  They  are  often 
called  metachromatic  granules  (vide  Fig.  1,  No.  16)  from  the  fact  that 
by  appropriate  procedure  they  can  be  stained  with  one  dye,  and  the 
protoplasm  in  which  they  lie  with  another  ;  sometimes,  when  a  single 
stain  is  used,  such  as  methylene  blue,  they  assume  a  slightly  different 
tint  from  the  protoplasm. 

For  the  demonstration  of  the  metachromatic  granules  two  methods 
have  been  advanced.  Ernst  recommends  that  a  few  drops  of  Loffler's 
methylene  blue  (vide  p.  98)  be  placed  on  a  cover-glass  preparation  and 
the  latter  passed  backwards  and  forwards  over  a  Bunsen  flame  for  half 
a  minute  after  steam  begins  to  rise.  The  preparation  is  then  washed 
in  water  and  counter-stained  for  one  to  two  minutes  in  watery  Bismarck- 
brown.  The  granules  are  here  stained  blue,  the  protoplasm  brown. 


STRUCTURE  OF  BACTERIAL  PROTOPLASM   9 

Neisser  stains  a  similar  preparation  in  warm  carbol-fuchsin,  washes 
with  1  per  cent  sulphuric  acid,  and  counter-stains  with  Lbffler's  blue. 
Here  the  granules  are  magenta,  the  protoplasm  blue.  The  general 
character  of  the  granules  thus  is  that  they  retain  the  first  stain  more 
intensely  than  the  rest  of  the  protoplasm  does. 

A  second  appearance  which  can  sometimes  be  seen  in  specimens 
stained  in  ordinary  ways  is  the  occurrence  of  a  concentration  of  the 
protoplasm  at  each  end  of  a  bacterium,  indicated  by  these  parts  being 
deeply  stained.  These  deeply  stained  parts  are  sometimes  called  polar 
granules  (vide  Fig.  1,  No.  16,  the  bacillus  most  to  the  right),  (German, 
Polkornchen  or  Polkoruer). 

With  regard  to  the  significance  that  is  to  be  attached  to  such 
appearances,  much  depends  on  whether  they  are  constantly  present 
under  all  circumstances,  or  only  occasionally,  when  the  organism  is 
grown  in  special  media  or  under  special  growth  conditions.  Some 
bacteria,  however  stained,  show  evidence  of  having  the  protoplasm 
somewhat  granular,  e.g.  the  diphtheria  bacillus.  In  other  cases  this 
granular  condition  is  only  seen  when  the  organism  has  been  grown  under 
bad  conditions,  or  where  the  food  supply  is  becoming  exhausted.  Some 
have  thought  that  the  appearances  might  be  due  to  a  process  allied  to 
mitosis  and  might  signify  approaching  division,  but  of  this  there  is  no 
evidence. 

In  perfectly  healthy  and  young  bacteria,  appearances  of  granule 
formation  and  of  vacuolation  may  be  accidentally  produced  by  physical 
means  in  the  occurrence  of  wha"t  is  known  as  plasmolysis.  To  speak 
generally,  when  a  mass  of  protoplasm  surrounded  by  a  fairly  firm 
envelope  of  a  colloidal  nature  is  placed  in  a  solution  containing  salts  in 
greater  concentration  than  that  in  which  it  has  previously  been  living, 
then  by  a  process  of  osmosis  the  water  held  in  the  protoplasm  passes 
out  through  the  membrane,  and,  the  protoplasm  retracting  from  the 
latter,  the  appearance  of  vacuolation  is  presented.  Now  in  making  a 
dried  film  for  the  microscopic  examination  of  bacteria  the  conditions 
necessary  for  the  occurrence  of  this  process  may  be  produced,  and  the 
appearances  of  vacuolation  and,  in  certain  cases,  of  Polkorner  may  thus 
be  brought  about.  Plasmolysis  in  bacteria  has  been  extensively 
investigated,1  and  has  been  found  to  occur  in  some  species  more  readily 
than  in  others.  Furthermore  it  is  often  most  readily  observed  in  old  or 
otherwise  enfeebled  cultures. 

Blitschli,  from,  a  study  of  some  large  sulphur-containing  forms,  con- 
cludes that  the  greater  part  of  the  bacterial  cell  may  correspond  to  a 
nucleus,  and  that  this  is  surrounded  by  a  thin  layer  of  protoplasm  which 
in  the  smaller  bacteria  escapes  notice,  unless  when,  as  in  the  bacilli,  it 
can  be  made  out  at  the  ends  of  the  cells.  Fischer,  it  may  be  said,  looks 
on  the  appearances  seen  in  Biitschli's  preparations  as  due  to  plasmolysis. 

The  Chemical  Composition  of  Bacteria. — In  the  bodies  of 
bacteria  many  definite  substances  occur.  Some  bacteria  have 
been  described  as  containing  chlorophyll,  but  these  are  properly 
to  be  classed  with  the  schizophyceae.  Sulphur  is  found  in  some 
of  the  higher  forms,  and  starch  granules  are  also  described  as 
occurring.  Many  species  of  bacteria,  when  growing  in  masses, 

1  Consult  Fischer,  "  Untersuchungen  uber  Bakterien,"  Berlin,  1894; 
"  Ueber  den  Bau  der  Cyanophyceeu  und  Bakterien,"  Jena,  1897. 


10   GENERAL  MORPHOLOGY  AND  BIOLOGY 

are  brilliantly  coloured,  though  few  bacteria  associated  with  the 
production  of  disease  give  rise  to  pigments.  In  some  of  the 
organisms  classed  as  bacteria  a  pigment  named  bacterio-purpurin 
has  been  observed  in  the  protoplasm,  and  similar  intracellular 
pigments  probably  occur  in  some  of  the  larger  forms  of  the 
lower  bacteria  and  may  occur  in  the  smaller ;  but  it  is  usually 
impossible  to  determine  whether  the  pigment  occurs  inside  or 
outside  the  protoplasm.  In  many  cases,  for  the  free  production 
of  pigment  abundant  oxygen  supply  is  necessary;  but  sometimes, 
as  in  the  case  of  spirillum  rubrum,  the  pigment  is  best  formed 
in  the  absence  of  oxygen.  Sometimes  the  faculty  of  forming  it 
may  be  lost  by  an  organism  for  a  time,  if  not  permanently,  by 
the  conditions  of  its  growth  being  altered.  Thus,  for  example, 
if  the  b.  pyocyaneus  be  exposed  to  the  temperature  of  42°  C. 
for  a  certain  time,  it  loses  its  power  of  producing  its  bluish 
pigment.  Pigments  formed  by  bacteria  often  diffuse  out  into, 
and  colour,  the  medium  for  a  considerable  distance  around. 

Comparatively  little  is  known  of  the  nature  of  bacterial  pigments. 
Zopf,  however,  has  found  that  many  of  them  belong  to  a  group  of 
colouring  matters  which  occur  widely  in  the  vegetable  and  animal 
kingdoms,  viz.  the  lipochromes.  These  lipochromes,  which  get  their 
name  from  the  colouring  matter  of  animal  fat,  include  the  colouring 
matter  in  the  petals  of  Ranunculacege,  the  yellow  pigments  of  serum  and 
of  the  yolks  of  eggs,  and  many  bacterial  pigments.  The  lipochromes  are 
characterised  by  their  solubility  in  chloroform,  alcohol,  ether,  and 
petroleum,  and  by  their  giving  indigo-blue  crystals  with  strong  sulphuric 
acid,  and  a  green  colour  with  iodine  dissolved  in  potassium  iodide. 
Though  crystalline  compounds  of  these  have  been  obtained,  their 
chemical  constitution  is  entirely  unknown  and  even  their  percentage 
composition  is  disputed. 

Some  observations  have  been  made  on  the  chemical  structure 
of  bacterial  protoplasm.  Nencki  isolated  from  the  bodies  of 
certain  putrefactive  bacteria  proteid  bodies  which,  according  to 
Ruppel,  appear  to  have  been  allied  to  peptone,  and  which 
differed  from  nucleo- proteids  in  not  containing  'phosphorus, 
but  many  of  the  proteids  isolated  by  other  chemists  have 
been  allied  in  their  nature  to  the  protoplasm  of  the  nuclei 
of  cells.  Buchner  in  certain  researches  obtained  bodies  of  this 
nature  allied  to  the  vegetable  caseins,  and  he  adduces  evidence 
to  show  that  it  is  to  these  that  the  characteristic  staining 
properties  are  due.  Various  observers  have  isolated  similar 
phosphorus-containing  proteids  from  different  bacteria.  Besides 
proteids,  however,  substances  of  a  different  nature  have  been 
isolated.  Thus  cellulose,  fatty  material,  chitin,  wax-like  bodies, 
and  other  substances  have  been  observed.  There  are  also  found 


THE   CLASSIFICATION   OF   BACTERIA  11 

various  mineral  salts,  especially  those  of  sodium,  potassium,  and 
magnesium.  The  amount  of  different  constituents  varies  ac- 
cording to  the  age  of  the  culture  and  the  medium  used  for 
growth,  and  certainly  great  variation  takes  place  in  the  com- 
position of  different  species. 

The  Classification  of  Bacteria. — There  have  been  numerous 
schemes  set  forth  for  the  classification  of  bacteria,  the  funda- 
mental principle  running  through  all  of  which  has  been  the 
recognition  of  the  two  sub-groups  and  the  type  forms  mentioned 
in  the  opening  paragraph  above.  In  the  attempts  to  still 
further  subdivide  the  group,  scarcely  two  systematists  are  agreed 
as  to  the  characters  on  which  sub-classes  are  to  be  based*.  Our 
present  knowledge  of  the  essential  morphology  and  relations  of 
bacteria  is  as  yet  too  limited  for  a  really  natural  classification  to 
be  attempted.  To  prepare  for  the  elaboration  of  the  latter, 
Marshall  Ward  suggested  that  in  every  species  there  should  be 
studied  the  habitat,  best  food  supply,  condition  as  to  gaseous 
environment,  range  of  growth,  temperature,  morphology,  life 
history,  special  properties  and  pathogenicity. 

We  must  thus  beacon  tent  with  a  provisional  and  incomplete 
classification.  We  have  said  that  the  division  into  lower  and 
higher  bacteria  is  recognised  by  all,  though,  as  in  every  other 
classification,  there  occur  transitional  forms.  In  subdividing 
the  bacteria  further,  the  forms  they  assume  constitute  at  present 
the  only  practicable  basis  of  classification.  The  lower  bacteria 
thus  naturally  fall  into  the  three  groups  mentioned,  the  cocci, 
bacilli,  and  spirilla,  though  the  higher  are  more  difficult  to  deal 
with.  Subsidiary,  though  important,  points  in  still  further  sub- 
division are  the  planes  in  which  fission  takes  place  and  the 
presence  or  absence  of  spores.  The  recognition  of  actual  species 
is  often  a  matter  of  great  difficulty.  The  points  to  be  observed 
in  this  will  be  discussed  later  (p.  115). 

I.  The  Lower  Bacteria.1 — These,  as  we  have  seen,  are 
minute  unicellular  masses  of  protoplasm  surrounded  by  an 
envelope,  the  total  vital  capacities  of  a  species  being  represented 
in  every  cell.  They  present  three  distinct  type  forms,  the 
coccus,  the  bacillus,  and  the  spirillum ;  endogenous  sporulation 
may  occur.  They  may  also  be  motile. 

1.  The  Cocci. — In  this  group  the  cells  range  in  different 
species  from  '5  //,  to  2  /x  in  diameter,  but  most  measure  about  1  ya. 
Before  division  they  may  increase  in  size  in  all  directions.  The 
species  are  usually  classified  according  to  the  method  of  division. 

1  For  the  illustration  of  this  and  the  succeeding  systematic  paragraphs, 
vide  Fig.  1. 


12       GENERAL   MORPHOLOGY   AND   BIOLOGY 

If  the  cells  divide  only  in  one  axis,  and  through  the  consistency 
of  their  envelopes  remain  attached,  then  a  chain  of  cocci  will  be 
formed.  A  species  in  which  this  occurs  is  known  as  a  strepto- 
coccus. If  division  takes  place  irregularly  the  resultant  mass  may 
be  compared  to  a  bunch  of  grapes,  and  the  species  is  often  called 
a  staphylococcus.  Division  may  take  place  in  two  axes  at  right 
angles  to  one  another,  in  which  case  cocci  adherent  to  each  other 
in  packets  of  four  (called  tetrads}  or  sixteen  may  be  found, 
the  former  number  being  the  more  frequent.  To  all  these  forms 
the  word  micrococcus  is  often  generally  applied.  The  individuals 
in  a  growth  of  micrococci  often  show  a  tendency  to  remain 
united  in  twos.  These  are  spoken  of  as  diplococci,  but  this  is 
not  a  distinctive  character,  since  every  coccus  as  a  result  of 
division  becomes  a  diplococcus,  though  in  some  species  the 
tendency  to  remain  in  pairs  is  well  marked.  The  adhesion  of 
cocci  to  one  another  depends  on  the  character  of  the  capsule. 
Often  this  has  a  well-marked  outer  limit  (micrococcus  tetragenus), 
sometimes  it  is  of  great  extent,  its  diameter  being  many  times 
that  of  the  coccus  (streptococcus  mesenteriodes).  It  is  especially 
among  the  streptococci  and  staphylococci  that  the  phenomenon 
of  the  formation  of  arthrospores  is  said  to  occur.  In  none  of 
the  cocci  have  endogenous  spores  been  certainly  observed.  The 
number  of  species  of  the  streptococci  and  staphylococci  probably 
exceeds  150.  Usually  included  in  this  group  are  coccus-like 
organisms  which  divide  in  three  axes  at  right  angles  to  one 
another.  These  are  usually  referred  to  as  sarcince.  If  the  cells 
are  lying  single  they  are  round,  but  usually  they  are  seen  in 
cubes  of  eight  with  the  sides  which  are  in  contact  slightly 
flattened.  Large  numbers  of  such  cubes  may  be  lying  together. 
The  sarcinae  are,  as  a  rule,  rather  larger  than  the  other  members 
of  the  group.  Most  of  the  cocci  are  non-motile,  but  a  few  motile 
species  possessing  flagella  have  been  described. 

2.  Bacilli. — These  consist  of  long  or  short  cylindrical  cells, 
with  rounded  or  sharply  rectangular  ends,  usually  not  more  than 
1  fj,  broad,  but  varying  very  greatly  in  length.  They  may  be 
motile  or  non-motile.  Where  flagella  occur,  these  may  be 
distributed  all  round  the  organism,  or  only  at  one  or  both  of 
the  poles  (pseudomonas).  Several  species  are  provided  with 
sharply -marked  capsules  (b.  pneumoniae).  In  many  species 
endogenous  sporulation  occurs.  The  spores  may  be  central  or 
terminal,  round,  oval,  or  spindle-shaped. 

Great  confusion  in  nomenclature  has  arisen  in  this  group  in  con- 
sequence of  the  different  artiticial  meanings  assigned  to  the  essentially 
synonymous  terms  bacterium  and  bacillus.  Migula,  for  instance,  applies 


THE   LOWER   BACTERIA 


13 


156 


FIG.  1.— 1.  Coccus.  2.  Streptococcus.  3.  Staphylococcus.  4.  Capsulatecl  diplococcus. 
5.  "  Biscuit  "-shaped  coccus.  6.  Tetrads.  7.  Sarcina  form.  8.  Types  of  bacilli 
(1-8  are  diagrammatic).  9.  Non-septate  spirillum  xlOOO.  10.  Ordinary  spirillum — 
(a)  comma-shaped  element;  (b)  formation  of  spiral  by  comma-shaped  elements 
XlOOO.  11.  Types  of  spore  formation.  12.  Flagellated  bacteria.  13.  Changes  in 
bacteria  produced  by  plasmolysis  (after  Fischer).  14.  Bacilli  with  terminal  proto- 
plasm (Biitschli).  15.  (a)  Bacillus  composed  of  five  protoplasmic  meshes  ;  (&)  proto- 
plasmic network  in  micrococcus  (Biitschli).  16.  Bacteria  containing  metachromatic 
granules  (Ernst,  Neisser) — some  contain  polar  granules.  17.  Beggiatoa  alba.  Both 
filaments  contain  sulphur  granules — one  is  septate.  18.  Thiothrix  tenuis  (Wino- 
gradski).  19.  'Leptothrix  innominata  (Miller).  20.  Cladothrix  clichotoma  (Zopt). 
21.  Streptothrixactinomyces  (Bostrom),  (a)  colony  under  low  power  ;  (6)  filament 
showing  true  branching ;  (c)  filament  containing  coccus-like  bodies  ;  (d)  filament 
with  club  at  end. 


14       GENERAL   MORPHOLOGY   AND   BIOLOGY 

the  former  term  to  non-motile  species,  the  latter  to  the  motile.  Hueppe, 
on  the  other  hand,  calls  those  in  which  endogenous  sporulation  does 
not  occur,  bacteria,  and  those  where  it  does,  bacilli.  In  the  ordinary 
terminology  of  systematic  bacteriology  the  word  bacterium  has  been 
almost  dropped,  and  is  reserved,  as  we  have  done,  as  a  general  term  for 
the  whole  group.  It  is  usual  to  call  all  the  rod-shaped  varieties  bacilli. 

3.  Spirilla. — These  consist  of  cylindrical  cells  more  or  less 
spiral  or  wavy.  Of  such  there  are  two  main  types.  In  one  there 
is  a  long  non-septate,  usually  slender,  wavy  or  spiral  thread 
(Fig.  1,  No.  9).  In  the  other  type  the  unit  is  a  short  curved 
rod  (often  referred  to  as  of  a  " comma"  shape).  When  two 
or  more  of  the  latter  occur,  as  they  often  do,  end  to  end 
with  their  curves  alternating,  then  a  wavy  or  spiral  thread 
results.  An  example  of  this  is  the  cholera  microbe  (Fig.  1, 
No.  10).  This  latter  type  is  of  much  more  frequent  occurrence, 
and  contains  the  more  important  species.  Among  the  first  group 
motility  is  often  not  associated,  as  far  as  is  known,  with  the 
possession  of  flagella.  The  cells  here  apparently  move  by  an 
undulating  or  screw-like  contraction  of  the  protoplasm.  Most  of 
the  motile  spirilla,  however,  possess  flagella.  Of  the  latter  there 
may  be  one  or  two,  or  a  bunch  containing  as  many  as  twenty,  at 
one  or  both  poles.  Division  takes  place  as  among  the  bacilli, 
and  in  some  species  endogenous  sporulation  has  been  observed. 

Three  terms  are  used  in  dividing  this  group,  to  which  different  authors 
have  given  different  meanings.  These  terms  are  spirillum,  spirochsete, 
vibrio.  Migula  makes  "  vibrio  "  synonymous  with  "microspira,"  which 
he  applies  to  members  of  the  group  which  possess  only  one  or  two  polar 
flagella  ;  "spirillum"  he  applies  to  similar  species  which  have  bunches 
of  polar  flagella,  while  "  spirochfete  "  is  reserved  for  the  long  unflagellated 
spiral  cells.  Hueppe  applies  the  term  "  spirochsete  "  to  forms  without 
endospores,  "vibrio"  to  those  with  endospores  in  which  during  sporula- 
tion the  organism  changes  its  form,  and  "spirillum"  to  the  latter 
when  no  change  of  form  takes  place  in  sporulation.  Flugge,  another 
systematist,  applies  "spirochsete"  and  " spirillum  "  indiscriminately  to 
any  wavy  or  corkscrew  form,  and  "vibrio"  to  forms  where  the  undula- 
tions are  not  so  well  marked.  It  is  thus  necessary,  in  denominating  such 
a  bacterium  by  a  specific  name,  to  give  the  authority  from  whom  the 
name  is  taken. 

Quite  recently  great  doubt  has  arisen  as  to  whether  many  of 
the  non-septate  spirillary  forms  are  to  be  looked  on  as  bacteria 
at  all, — the  view  being  taken  that  in,  it  may  be,  many  cases 
they  represent  a  stage  in  the  life  history  of  what  are  really 
protozoa  of  the  nature  of  trypanosomes.  The  ultimate  classifica- 
tion of  the  spirilla  must  thus  be  left  an  open  question. 

II.  The  Higher  Bacteria. — These  show  advance  on  the  lower 
in  consisting  of  definite  filaments  branched  or  unbranched.  In 


THE   HIGHER   BACTEEIA  15 

most  cases  the  filaments  at  more  or  less  regular  intervals  are  cut 
by  septa  into  short  rod-shaped  or  curved  elements.  Such 
elements  are  more  or  less  interdependent  on  one  another,  and 
special  staining  methods  are  often  necessary  to  demonstrate  the 
septa  which  demarcate  the  individuals  of  a  filament.  There  is 
further  often  a  definite  membrane  or  sheath  common  to  all  the 
elements  in  a  filament.  Not  only,  however,  is  there  this  close 
organic  relationship  between  the  elements  of  the  higher  bacteria, 
but  there  is  also  interdependence  of  function ;  for  example,  one 
end  of  a  filament  is  frequently  concerned  merely  in  attaching 
the  organism  to  some  other  object.  The  greatest  advance, 
however,  consists  in  the  setting  apart  among  most  of  the  higher 
bacteria  of  the  free  terminations  of  the  filaments  for  the  produc- 
tion of  new  individuals,  as  has  been  described  (p.  2).  There 
are  various  classes  under  which  the  species  of  the  higher  bacteria 
are  grouped ;  but  our  knowledge  of  them  is  still  somewhat 
limited,  as  many  of  the  members  have  not  yet  been  artificially 
cultivated.  The  beggiatoa  group  consists  of  free  swimming 
forms,  motile  by  undulating  contractions  of  their  protoplasm. 
For  the  demonstration  of  the  rod-like  elements  of  the  filaments 
special  staining  is  necessary.  The  filaments  have  no  special 
sheath,  and  the  protoplasm  contains  sulphur  granules.  The 
method  of  reproduction  is  doubtful.  The  thiothrix  group  re- 
sembles the  last  in  structure,  and  the  protoplasm  also  contains 
sulphur  granules;  but  the  filaments  are  attached  at  one  end, 
and  at  the  other  form  gonidia.  The  leptothrix  group  resembles 
closely  the  thiothrix  group,  but  the  protoplasm  does  not  contain 
sulphur  granules.  In  the  cladothrix  group  there  is  the  appearance 
of  branching,  which,  however,  is  of  a  false  kind.  What  happens 
is  that  a  terminal  cell  divides,  and  on  dividing  again,  it  pushes 
the  product  of  its  first  division  to  one  side.  There  are  thus  two 
terminal  cells  lying  side  by  side,  and  as  each  goes  on  dividing, 
the  appearance  of  branching  is  given.  Here,  again,  there  is 
gonidium  formation ;  and  while  the  parent  organism  is  in  some 
of  its  elements  motile,  the  gonidia  move  by  means  of  flagella. 
The  highest  development  is  in  the  streptothrix  group,  to  which 
belongs  the  streptothrix  actinomyces,  or  the  actinomyces  bo  vis,  and 
several  other  important  pathogenic  agents.  Here  the  organism 
consists  of  a  felted  mass  of  non-septate  filaments,  in  which  true 
dichotomous  branching  occurs.  Under  certain  circumstances 
threads  grow  out,  and  produce  chains  of  coccus-like  bodies  from 
which  new  individuals  can  be  reproduced.  Such  bodies  are  often 
referred  to  as  spores,  but  they  have  not  the  same  staining  reactions 
nor  resisting  powers  of  so  high  a  degree  as  ordinary  bacterial 


16        GENERAL   MORPHOLOGY  AND   BIOLOGY 

spores.  Sometimes  too  the  protoplasm  of  the  filaments  breaks 
up  into  bacillus-like  elements,  which  may  also  have  the  capacity 
of  originating  new  individuals.  In  the  streptothrix  actinomyces 
there  may  appear  a  club-shaped  swelling  of  the  membrane  at  the 
end  of  the  filament,  which  has  by  some  been  looked  on  as  an 
organ  of  fructification,  but  which  is  most  probably  a  product  of 
a  degenerative  change.  The  streptothrix  group,  though  its 
morphology  and  relationships  are  much  disputed,  may  be  looked 
on  as  a  link  between  the  bacteria  on  the  one  hand,  and  the 
lower  fungi  on  the  other.  Like  the  latter,  the  streptothrix  forms 
show  the  felted  mass  of  non-septate  branching  filaments,  which 
is  usually  called  a  mycelium.  On  the  other  hand,  the  breaking 
up  of  the  protoplasm  of  the  streptothrix  into  coccus-  and  bacillus- 
like  forms,  links  it  to  the  other  bacteria. 

GENERAL  BIOLOGY  OF  THE  BACTERIA. 

There  are  five  prime  factors  which  must  be  considered  in 
the  growth  of  bacteria,  namely,  food  supply,  moisture,  relation  to 
gaseous  environment,  temperature,  and  light. 

Food  Supply. — The  bacteria  are  chiefly  found  living  on  the 
complicated  organic  substances  which  form  the  bodies  of  dead 
plants  and  animals,  or  which  are  excreted  by  the  latter  while 
they  are  yet  alive.  Seeing  that,  as  a  general  rule,  many  bacteria 
grow  side  by  side,  the  food  supply  of  any  particular  variety  is, 
relatively  to  it,  altered  by  the  growth  of  the  other  varieties 
present.  It  is  thus  impossible  to  imitate  the  complexity  of  the 
natural  food  environment  of  any  species.  The  artificial  media 
used  in  bacteriological  work  may  therefore  be  poor  substitutes 
for  the  natural  supply.  In  certain  cases,  however,  the  conditions 
under  which  we  grow  cultures  may  be  better  than  the  natural 
conditions.  For  while  one  of  two  species  of  bacteria  growing 
side  by  side  may  favour  the  growth  of  the  other,  it  may  also 
in  certain  cases  hinder  it,  and,  therefore,  when  the  latter  is 
grown  alone  it  may  grow  better.  Most  bacteria  seem  to 
produce  excretions  which  are  unfavourable  to  their  own 
vitality,  for,  when  a  species  is  sown  on  a  mass  of  artificial 
food  medium,  it  does  not  in  the  great  majority  of  cases  go  on 
growing  till  the  food  supply  is  exhausted,  but  soon  ceases  to 
grow.  Effete  products  diffuse  out  into  the  medium  and  prevent 
growth.  Such  diffusion  may  be  seen  when  the  organism  pro- 
duces pigment,  e.g.  b.  pyocyaneus  growing  on  gelatin.  In 
supplying  artificial  food  for  bacterial  growth,  the  general  principle 
ought  to  be  to  imitate  as  nearly  as  possible  the  natural  surround- 


RELATION   TO   GASEOUS   ENVIRONMENT       17 

ings,  though  it  is  found  that  there  exists  a  considerable  adapta- 
bility among  organisms.  With  the  pathogenic  varieties  it  is 
usually  found  expedient  to  use  media  derived  from  the  fluids  of 
the  animal  body,  and  in  cases  where  bacteria  growing  on  plants 
are  being  studied,  infusions  of  the  plants  on  which  they  grow 
are  frequently  used.  Some  bacteria  can  exist  on  inorganic  food, 
but  most  require  organic  material  to  be  supplied.  Of  the  latter, 
some  require  for  their  proper  nourishment  proteid  to  be  present, 
while  others  can  derive  their  nitrogen  from  such  a  non-proteid 
as  asparagin.  All  bacteria  require  nitrogen  to  be  present  in 
some  form,  and  many  require  to  derive  their  carbon  from 
carbohydrates.  Mineral  salts,  especially  sulphates,  chlorides,  and 
phosphates,  and  also  salts  of  iron  are  necessary.  Occasionally 
special  substances  are  needed  to  support  life.  Thus  some 
species,  in  the  protoplasm  of  which  sulphur  granules  occur, 
require  sulphuretted  hydrogen  to  be  present.  In  nature  the 
latter  is  usually  provided  by  the  growth  of  other  bacteria.  When 
the  food  supply  of  a  bacterium  fails,  it  degenerates  and  dies. 
The  proof  of  death  lies  in  the  fact  that  when  it  is  transferred  to 
fresh  and  good  food  supply  it  does  not  multiply.  If  the 
bacterium  spores,  it  may  then  survive  the  want  of  food  for  a 
very  long  time.  It  may  here  be  stated  that  the  reaction  of  the 
food  medium  is  a  matter  of  great  importance.  Most  bacteria 
prefer  a  slightly  alkaline  medium,  and  some,  e.g.  the  cholera 
spirillum,  will  not  grow  in  the  presence  of  the  smallest  amount 
of  free  acid. 

Moisture. — The  presence  of  water  is  necessary  for  the  con- 
tinued growth  of  all  bacteria.  The  amount  of  drying  which 
bacteria  in  the  vegetative  stage  will  resist  varies  very  much  in 
different  species.  Thus  the  cholera  spirillum  is  killed  by  two  or 
three  hours'  drying,  while  the  staphylococcus  pyogenes  aureus 
will  survive  ten  days'  drying,  and  the  bacillus  diphtherise  still 
more.  In  the  case  of  spores  the  periods  are  much  longer. 
Anthrax  spores  will  survive  drying  for  several  years,  but  here 
again  moisture  enables  them  to  resist  longer  than  when  they  are 
quite  dry.  When  organisms  have  been  subjected  to  such  hostile 
influences,  even  though  they  survive,  it  by  no  means  follows  that 
they  retain  all  their  vital  properties. 

Relation  to  Gaseous  Environment. — The  relation  of  bacteria 
to  the  oxygen  of  the  air  is  such  an  important  factor  in  the  life 
of  bacteria  that  it  enables  a  biological  division  to  be  made  among 
them.  Some  bacteria  will  only  live  and  grow  when  oxygen  is 
present.  To  these  the  title  of  obligatory  aerobes  is  given.  Other 
bacteria  will  only  grow  when  no  oxygen  is  present.  These  are 
2 


18       GENERAL   MORPHOLOGY   AND   BIOLOGY 

called  obligatory  anaerobes.  In  still  other  bacteria  the  presence 
or  absence  of  oxygen  is  a  matter  of  indifference.  This  group 
might  theoretically  be  divided  into  those  which  are  preferably 
aerobes,  but  can  be  anaerobes,  and  those  which  are  preferably 
anaerobes,  but  can  be  aerobes.  As  a  matter  of  fact  such 
differences  are  manifested  to  a  slight  degree,  but  all  such 
organisms  are  usually  grouped  as  facultative  anaerobes,  i.e.  pre- 
ferably aerobic  but  capable  of  existing  without  oxygen.  Examples 
of  obligatory  aerobes  are  b.  proteus  vulgaris,  b.  subtilis;  of 
obligatory  anaerobes,  b.  tetani,  b.  oedematis  maligni,  while  the 
great  majority  of  pathogenic  bacteria  are  facultative  anaerobes. 
With  regard  to  anaerobes,  hydrogen  and  nitrogen  are  indifferent 
gases.  Many  anaerobes,  however,  do  not  flourish  well  in  an 
atmosphere  of  carbon  dioxide.  Very  few  experiments  have 
been  made  to  investigate  the  action  on  bacteria  of  gas  under 
pressure.  A  great  pressure  of  carbon  dioxide  is  said  to  make 
the  b.  anthracis  lose  its  power  of  sporing,  but  it  seems  to  have 
no  effect  on  its  vitality  or  on  that  of  the  b.  typhosus.  With 
the  bacillus  pyocyaneus,  however,  it  is  said  to  destroy  life. 

Temperature. — For  every  species  of  bacterium  there  is  a 
temperature  at  which  it  grows  best.  This  is  called  the  "  optimum 
temperature."  There  is  also  in  each  case  a  maximum  tempera- 
ture above  which  growth  does  not  take  place,  and  a  minimum 
temperature  below  which  growth  does  not  take  place.  As  a 
general  rule  the  optimum  temperature  is  about  the  temperature 
of  the  natural  habitat  of  the  organism.  For  organisms  taking 
part  in  the  ordinary  processes  of  putrefaction  the  temperature  of 
warm  summer  weather  (20°  to  24°  C.)  may  be  taken  as  the 
average  optimum,  while  for  organisms  normally  inhabiting  animal 
tissues  35°  to  39°  C  is  a  fair  average.  The  lowest  limit  of 
ordinary  growth  is  from  12°  to  14°  C.,  and  the  upper  is  from 
42°  to  44°  C.  In  exceptional  cases  growth  may  take  place  as 
low  as  5°  C.,  and  as  high  as  70°  C.  Some  organisms  which 
grow  best  at  a  temperature  of  from  60°  to  70°  C.  have  been 
isolated  from  dung,  the  intestinal  tract,  etc.  These  have  been 
called  thermophilic  bacteria.  It  is  to  be  noted  that  while  growth 
does  not  take  place  below  or  above  a  certain  limit  it  by 
no  means  follows  that  death  takes  place  outside  such  limits. 
Organisms  can  resist  cooling  below  their  minimum  or  heating 
beyond  their  maximum  without  being  killed.  Their  vital  activity 
is  merely  paralysed.  Especially  is  this  true  of  the  effect  of  cold 
on  bacteria.  The  results  of  different  observers  vary ;  but  if  we 
take  as  an  example  the  cholera  vibrio,  Koch  found  that  while 
the  minimum  temperature  of  growth  was  16°  C.,  a  culture  might 


CONDITIONS  AFFECTING  BACTERIAL  MOTILITY    19 

be  cooled  to  -32°  C.  without  being  killed.  With  regard  to  the 
upper  limit,  few  ordinary  organisms  in  a  spore-free  condition  will 
survive  a  temperature  of  57°  C.,  if  long  enough  applied.  Many 
organisms  lose  some  of  their  properties  when  grown  at  unnatural 
temperatures.  Thus  many  pathogenic  organisms  lose  their 
virulence  if  grown  above  their  optimum  temperature,  and  some 
chromogenic  forms,  most  of  which  prefer  rather  low  tempera- 
tures, lose  their  capacity  of  producing  pigment,  e.g.  spirillum 
rubrum. 

Effect  of  Light. — Of  recent  years  much  attention  has  been 
paid  to  this  factor  in  the  life  of  bacteria.  Direct  sunlight  is 
found  to  have  a  very  inimical  effect.  It  has  been  found  that 
an  exposure  of  dry  anthrax  spores  for  one  and  a  half  hours  to 
sunlight  kills  them.  When  they  are  moist,  a  much  longer 
exposure  is  necessary.  Typhoid  bacilli  are  killed  in  about 
one  and  a  half  hours,  and  similar  results  have  been  obtained 
with  many  other  organisms.  In  such  experiments  the  thickness 
of  the  medium  surrounding  the  growth  is  an  important  point. 
Death  takes  place  more  readily  if  the  medium  is  scanty  or  if  the 
organisms  are  suspended  in  water.  Any  fallacy  which  might 
arise  from  the  effect  of  the  heat  rays  of  the  sun  has  been  ex- 
cluded, though  light  plus  heat  is  more  fatal  than  light  alone. 
In  direct  sunlight  it  is  chiefly  the  green,  violet,  and,  it  may  be, 
the  ultra-violet  rays  which  are  fatal.  Diffuse  daylight  has  also  a 
bad  effect  upon  bacteria,  though  it  takes  a  much  longer  exposure 
to  do  serious  harm.  A  powerful  electric  light  is  as  fatal  as  sun- 
light. Here,  as  with  other  factors,  the  results  vary  very  much 
with  the  species  under  observation,  and  a  distinction  must  be 
drawn  between  a  mere  cessation  of  growth  and  the  condition  of 
actual  death.  Some  bacteria  especially  occurring  on  the  dead 
bodies  of  fresh  fish  are  phosphorescent. 

Conditions  affecting  the  Movements  of  Bacteria. — In  some 
cases  differences  are  observed  in  the  behaviour  of  motile  bacteria, 
contemporaneous  with  changes  in  their  life  history.  Thus,  in 
the  case  of  bacillus  subtilis,  movement  ceases  when  sporulation 
is  about  to  take  place.  On  the  other  hand,  in  the  bacillus  of 
symptomatic  anthrax,  movement  continues  while  sporulation  is 
progressing.  Under  ordinary  circumstances  motile  bacteria 
appear  not  to  be  constantly  moving  but  occasionally  to  rest. 
In  every  case  the  movements  become  more  active  if  the 
temperature  be  raised.  Most  interest,  however,  attaches  to  the 
fact  that  bacilli  may  be  attracted  to  certain  substances  and 
repelled  by  others.  Schenk,  for  instance,  observed  that  motile 
bacteria  were  attracted  to  a  warm  point  in  a  way  which  did  not 


20       GENERAL   MORPHOLOGY   AND   BIOLOGY 

occur  when  the  bacteria  were  dead  and  therefore  only  subject 
to  physical  conditions.  Most  important  observations  have  been 
made  on  the  attraction  and  repulsion  exercised  on  bacteria  by 
chemical  agents,  which  have  been  denominated  respectively 
positive  and  negative  ckemiotaxis.  Pfeffer  investigated  this 
subject  in  many  lowly  organisms,  including  bacterium  termo 
and  spirillum  undula.  The  method  was  to  fill  with  the  agent 
a  fine  capillary  tube,  closed  at  one  end,  to  introduce  this  into  a 
drop  of  fluid  containing  the  bacteria  under  a  cover-glass,  and 
to  watch  the  effect  through  the  microscope.  The  general  result 
was  to  indicate  that  motile  bacteria  may  be  either  attracted  or 
repelled  by  the  fluid  in  the  tube.  The  effect  of  a  given  fluid 
differs  in  different  organisms,  and  a  fluid  chemiotactic  for  one 
organism  may  not  act  on  another.  Degree  of  concentration  is 
important,  but  the  nature  of  the  fluid  is  more  so.  Of  inorganic 
bodies  salts  of  potassium  are  the  most  powerfully  attracting 
bodies,  and  in  comparing  organic  bodies  the  important  factor 
is  the  molecular  constitution.  These  observations  have  been 
confirmed  by  Ali-Cohen,  who  found  that  while  the  vibrio  of 
cholera  and  the  typhoid  bacillus  were  scarcely  attracted  by 
chloride  of  potassium,  they  were  powerfully  influenced  by 
potato  juice.  Further,  the  filtered  products  of  the  growth  of 
many  bacteria  have  been  found  to  have  powerful  chemiotactic 
properties.  It  is  evident  that  all  these  observations  have  a 
most  important  bearing  on  the  action  of  bacteria,  though  we  do 
not  yet  know  their  true  significance.  Corresponding  chemio- 
tactic phenomena  are  shown  also  by  certain  animal  cells,  e.g. 
leucocytes,  to  which  reference  is  made  below. 

The  Parts  played  by  Bacteria  in  Nature. — As  has  been  said, 
the  chief  effect  of  bacterial  action  in  nature  is  to  break  up  into 
more  simple  combinations  the  complex  molecules  of  the  organic 
substances  which  form  the  bodies  of  plants  and  animals,  or 
which  are  excreted  by  them.  In  some  cases  we  know  some  of 
the  stages  of  disintegration,  but  in  most  cases  we  know  only 
general  principles  and  sometimes  only  results.  In  the  case  of 
milk,  for  instance,  we  know  that  lactic  acid  is  produced  from 
the  lactose  by  the  action  of  the  bacillus  acidi  lactici  and  of 
other  bacteria,  and  that  from  urea  ammonium  carbonate 
is  produced  by  the  micrococcus  ureae.  That  the  very  compli- 
cated process  of  putrefaction  is  due  to  bacteria  is  absolutely 
proved,  for  any  organic  substance  can  be  preserved  indefinitely 
from  ordinary  putrefaction  by  the  adoption  of  some  method  of 
killing  all  bacteria  present  in  it,  as  will  be  afterwards  described. 
This  statement,  however,  does  not  exclude  the  fact  that  molecular 


ACTION   OF   BACTEKIAL   FERMENTS  21 

changes  take  place  spontaneously  in  the  passing  of  the  organic 
body  from  life  to  death.  Many  processes  not  usually  referred  to 
as  putrefactive  are  also  bacterial  in  their  origin.  The  souring 
of  milk,  already  referred  to,  the  becoming  rancid  of  butter,  the 
ripening  of  cream  and  of  cheese,  are  all  due  to  bacteria. 

A  certain  comparatively  small  number  of  bacteria  have  been 
proved  to  be  the  causal  agents  in  some  disease  processes 
occurring  in  man,  animals,  and  plants.  This  means  that  the 
fluids  and  tissues  of  living  bodies  are,  under  certain  circum- 
stances, a  suitable  pabulum  for  the  bacteria  involved.  The 
effects  of  the  action  of  these  bacteria  are  analogous  to  those 
taking  place  in  the  action  of  the  same  or  other  bacteria  on  dead 
animal  or  vegetable  matter.  The  complex  organic  molecules 
are  broken  up  into  simpler  products.  We  shall  study  these 
processes  more  in  detail  later.  Meantime  we  may  note  that 
the  disease -producing  effects  of  bacteria  form  the  basis  of 
another  .biological  division  of  the  group.  Some  bacteria  are 
harmless  to  animals  and  plants,  and  apparently  under  no 
circumstances  give  rise  to  disease  in  either.  These  are  known 
as  saprophytes.  They  are  normally  engaged  in  breaking  up 
dead  animal  and  vegetable  matter.  Others  normally  live  on 
or  in  the  bodies  of  plants  and  animals  and  produce  disease. 
These  are  known  as  parasitic  bacteria.  Sometimes  an  attempt 
is  made  to  draw  a  hard  and  fast  line  between  the  saprophytes 
and  the  parasites,  and  obligatory  saprophytes  or  parasites  are 
spoken  of.  This  is  an  erroneous  distinction.  Some  bacteria 
which  are  normally  saprophytes  can  produce  pathogenic  effects 
(e.g.  bacillus  oedematis  maligni),  and  it  is  consistent  with  our 
knowledge  that  the  best-known  parasites  may  have  been  derived 
from  saprophytes.  On  the  other  hand,  the  fact  that  most 
bacteria  associated  with  disease  processes,  and  proved  to  be 
the  cause  of  the  latter,  can  be  grown  in  artificial  media,  shows 
that  for  a  time  at  least  such  parasites  can  be  saprophytic.  As 
to  how  far  such  a  saprophytic  existence  of  disease-producing 
bacteria  occurs  in  nature,  we  are  in  many  instances  still  ignorant. 

The  Methods  of  Bacterial  Action. — The  processes  which 
bodies  undergo  in  being  split  up  by  bacteria  depend,  first,  on 
the  chemical  nature  of  the  bodies  involved  and,  secondly,  on  the 
varieties  of  the  bacteria  which  are  acting.  The  destruction  of 
albuminous  bodies  which  is  mostly  involved  in  the  wide  and 
varied  process  of  putrefaction  can  be  undertaken  by  whole 
groups  of  different  varieties  of  bacteria.  The  action  of  the 
latter  on  such  substances  is  analogous  to  what  takes  place  when 
albumins  are  subjected  to  ordinary  gastric  and  intestinal  digestion. 


22       GENERAL   MORPHOLOGY   AND   BIOLOGY 

In  these  circumstances,  therefore,  the  production  of  albumoses, 
peptones,  etc.,  similar  to  those  of  ordinary  digestion,  can  be 
recognised  in  putrefying  solutions,  though  the  process  of  destruc- 
tion always  goes  further,  and  still  simpler  substances,  e.g.  indol, 
and,  it  may  be,  crystalline  bodies  of  an  alkaloidal  nature,  are 
the  ultimate  results.  The  process  is  an  exceedingly  complicated 
one  when  it  takes  place  in  nature,  and  different  bacteria  are 
probably  concerned  in  the  different  stages.  Many  other  bacteria, 
e.g.  some  pathogenic  forms,  though  not  concerned  in  ordinary 
putrefactive  processes,  have  a  similar  digestive  capacity.  When 
carbohydrates  are  being  split  up,  then  various  alcohols,  ethers, 
and  acids  are  produced.  During  bacterial  growth  there  is 
not  infrequently  the  abundant  production  of  such  gases  as 
sulphuretted  hydrogen,  carbon  dioxide,  methane,  etc.  For  an 
exact  knowledge  of  the  destructive  capacities  of  any  particular 
bacterium  there  must  be  an  accurate  chemical  examination  of  its 
effects  when  it  has  been  grown  in  artificial  media  the  nature  of 
which  is  known.  The  precise  substances  it  is  capable  of  forming 
can  thus  be  found  out.  Many  substances,  however,  are  produced 
by  bacteria,  of  the  exact  nature  of  which  we  are  still  ignorant, 
for  example,  the  toxic  bodies  which  play  such  an  important  part 
in  the  action  of  many  pathogenic  species. 

Many  of  the  actions  of  bacteria  depend  on  the  production  by 
them  of  ferments  of  a  very  varied  nature  and  complicated  action. 
Thus  the  digestive  action  on  albumins  probably  depends  on  the 
production  of  a  peptic  ferment  analogous  to  that  produced  in 
the  animal  stomach.  Ferments  which  invert  sugar,  which  split 
sugars  up  into  alcohols  or  acids,  which  coagulate  casein,  which 
split  up  urea  into  ammonium  carbonate,  also  occur. 

Such  ferments  may  be  diffused  into  the  surrounding  fluid,  or 
be  retained  in  the  cells  where  they  are  formed.  Sometimes  the 
breaking  down  of  the  organic  matter  appears  to  take  place 
within,  or  in  the  immediate  proximity  of,  the  bacteria,  sometimes 
wherever  the  soluble  ferments  reach  the  organic  substances. 
And  in  certain  cases  the  ferments  diffused  out  into  the  sur- 
rounding medium  probably  break  down  the  constituents  of 
the  latter  to  some  extent,  and  prepare  them  for  a  further, 
probably  intracellular,  disintegration.  Thus  in  certain  putre- 
factions of  fibrin,  if  the  process  be  allowed  to  go  on  naturally, 
the  fibrin  dissolves  and  ultimately  great  gaseous  evolution 
of  carbon  dioxide  and  ammonia  takes  place,  but  if  the 
bacteria,  shortly  after  the  process  has  begun,  are  killed  or 
paralysed  by  chloroform,  then  only  a  peptonisation  of  the 
fibrin  occurs,  without  the  further  splitting  up  and  gaseous  pro- 


VARIABILITY   AMONG   BACTERIA  23 

duction  being  observed.  That  a  purely  intracellular  digestion 
may  take  place  is  .illustrated  by  what  has  been  shown  to  occur 
in  the  case  of  the  micrococcus  ureae,  which  from  urea  forms 
ammonium  carbonate  by  adding  water  to  the  urea  molecule. 
Here,  if  after  the  action  has  commenced,  the  bacteria  are  filtered 
off,  no  further  production  of  ammonium  carbonate  takes  place, 
which  shows  that  no  ferment  has  been  dissolved  out  into  the 
urine.  If  now  the  bodies  of  the  bacteria  be  extracted  with 
absolute  alcohol  or  ether,  which  of  course  destroy  their  vitality, 
a  substance  is  obtained  of  the  nature  of  a  ferment,  which,  when 
added  to  sterile  urine,  rapidly  causes  the  production  of  ammonium 
carbonate.  This  ferment  has  evidently  been  contained  within 
the  bacterial  cells. 

In  considering  the  effects  of  bacteria  in  nature  it  must  be  recognised 
that  some  species  are  capable  of  building  up  complex  substances  out  of 
simple  chemical  compounds.  Examples  of  these  are  found  in  the 
bacteria  which  in  the  soil  make  nitrogen  more  available  for  plant  nutri- 
tion by  converting  ammonia  into  nitrites  and  nitrates.  Winogradski,  by 
using  media  containing  non-nitrogenous  salts  of  magnesium,  potassium, 
and  ammonium,  and  free  of  organic  matter,  has  demonstrated  the 
existence  of  forms  which  convert,  by  oxidation,  ammonia  into  nitrites, 
and  of  other  forms  which  convert  these  nitrites  into  nitrates.  Both  can 
derive  their  necessary  carbon  from  alkaline  carbonates.  Other  bacteria, 
or  organisms  allied  to  the  bacteria,  exist  which  can  actually  take  up  and 
combine  into  new  compounds  the  free  nitrogen  of  the  air.  These  are 
found  in  the  tubercles  which  develop  on  the  rootlets  of  the  leguininosae. 
Without  such  organisms  the  tubercles  do  not  develop,  and  without  the 
development  of  the  tubercles  the  plants  are  poor  and  stunted.  Bacteria 
thus  play  an  important  part  in  the  enrichment  and  fertilisation  of  the 
soil. 

The  Occurrence  of  Variability  among  Bacteria. — The  question  of  the 
division  of  the  group  of  bacteria  into  definite  species  has  given  rise  to 
much  discussion  among  vegetable  and  animal  morpliologists,  and  at 
one  time  very  divergent  views  were  held.  Some  even  thought  that  the 
same  species  might  at  one  time  give  rise  to  one  disease, — at  another 
time  to  another.  There  is,  however,  now  practical  unanimity  that 
bacteria  show  as  distinct  species  as  the  other  lower  plants  and  animals, 
though,  of  course,  the  difficulty  of  defining  the  concept  of  a  species  is  as 
great  in  them  as  it  is  in  the  latter.  Still,  we  can  say  that  among  the 
bacteria  we  see  exhibited  (to  use  the  words  of  De  Bary)  "the  same 
periodically  repeated  course  of  development  within  certain  empirically 
determined  limits  of  variation  "  which  justifies,  among  higher  forms  of 
life,  a  species  to  be  recognised.  What  at  first  raised  doubts  as  to  the 
occurrence  of  species  among. the  bacteria  was  the  observation  in  certain 
cases  of  what  is  known  as  pleomorphism.  By  this  is  meant  that  one 
species  may  assume  at  different  times  different  forms,  e.g.  appear  as  a 
coccus,  a  bacillus,  or  a  leptothrix.  Undoubtedly,  many  of  the  cases 
where  this  was  alleged  to  have  been  observed  occurred  before  the 
elaboration  of  the  modern  technique  for  the  obtaining  of  pure  cultures, 
but  at  the  present  day  there  are  cases  where  evidence  appears  to  exist 


24       GENERAL   MORPHOLOGY  AND   BIOLOGY 

of  the  occurrence  of  pleomorphism.  This  is  especially  the  case  with 
certain  bacilli,  and  it  may  lead  to  such  forms  being  classed  among  the 
higher  bacteria.  Pleomorphism  is,  however,  a  rare  condition,  and  with 
regard  to  the  bacteria  as  a  whole  we  may  say  that  each  variety  tends  to 
conform  to  a  definite  type  of  structure  and  function  which  is  peculiar  to 
it  and  to  it  alone.  On  the  other  hand,  slight  variations  from  such  type 
can  occur  in  each.  The  size  may  vary  a  little  with  the  medium  in 
which  the  organism  is  growing,  and  under  certain  similar  conditions  the 
adhesion  of  bacteria  to  each  other  may  also  vary.  Thus  cocci,  which  are 
ordinarily  seen  in  short  chains,  may  grow  in  long  chains.  The  capacity 
to  form  spores  may  be  altered,  and  such  properties  as  the  elaboration 
of  certain  ferments  or  of  certain  pigments  may  be  impaired.  Also  the 
characters  of  the  growths  on  various  media  may  undergo  variations. 
As  has  been  remarked,  variation  as  observed  consists  largely  in  a  tendency 
in  a  bacterium  to  lose  properties  ordinarily  possessed,  and  all  attempts 
to  transform  one  bacterium  into  an  apparently  closely  allied  variety 
(such  as  the  b.  coli  into  the  b.  typhosus)  have  failed.  This  of  course 
does  not  preclude  the  possibility  of  one  species  having  been  originally 
derived  from  another  or  of  both  having  descended  from  a  common 
ancestor,  but  we  can  say  that  only  variations  of  an  unimportant  order 
have  been  observed  to  take  place,  and  here  it  must  be  remembered  that 
in  many  cases  we  can  have  forty-eight  or  more  generations  under  obser- 
vation within  twenty- four  hours. 


CHAPTEK  II. 

METHODS  OF  CULTIVATION  OF  BACTERIA. 

Introductory. — In  order  to  study  the  characters  of  any  species 
of  bacterium  it  is  necessary  to  have  it  growing  apart  from  every 
other  species.  In  the  great  majority  of  cases  where  bacteria 
occur  in  nature,  this  condition  is  not  fulfilled.  Only  in  the 
blood  and  tissues  in  some  diseases  do  particular  species  occur 
singly  and  alone.  We  usually  have,  therefore,  to  remove  a 
bacterium  from  its  natural  surroundings  and  grow  it  on  an 
artificial  food  medium.  When  we  have  succeeded  in  separating 
it,  and  have  got  it  to  grow  on  a  medium  which  suits  it,  we  are 
said  to  have  obtained  a  pure  culture.  The  recognition  of 
different  species  of  bacteria  depends,  in  fact,  far  more  on  the 
characters  presented  by  pure  cultures  and  their  behaviour  in 
different  food  media,  than  on  microscopic  examination.  The 
latter  in  most  cases  only  enables  us  to  refer  a  given  bacterium 
to  its  class.  Again,  in  inquiring  as  to  the  possible  possession 
of  pathogenic  properties  by  a  bacterium,  the  obtaining  of  pure 
cultures  is  absolutely  essential. 

To  obtain  pure  cultures,  then,  is  the  first  requisite  of 
bacteriological  research.  Now,  as  bacteria  are  practically 
omnipresent,  we  must  first  of  all  have  means  of  destroying  all 
extraneous  organisms  which  may  be  present  in  the  food  media 
to  be  used  in  the  vessels  in  which  the  food  media  are  contained, 
and  on  all  instruments  which  are  to  come  in  contact  with  our 
cultures.  The  technique  of  this  destructive  process  is  called 
sterilisation.  We  must  therefore  study  the  methods  of  sterilisation. 
The  growth  of  bacteria  in  other  than  their  natural  surroundings 
involves  further  the  preparation  of  sterile  artificial  food  media, 
and  when  we  have  such  media  prepared  we  have  still  to  look  at 
the  technique  of  the  separation  of  micro-organisms  from  mixtures 
of  these,  and  the  maintaining  of  pure  cultures  when  the  latter 
have  been  obtained.  We  shall  here  find  that  different  methods 

25 


26      METHODS   OF   CULTIVATION   OF   BACTERIA 

are  necessary  according  as  we  are  dealing  with  aerobes  or  anaerobes. 
Each  of  these  methods  will  be  considered  in  turn. 


THE   METHODS  OF  STERILISATION. 

To  exclude  extraneous  organisms,  all  food  materials,  glass 
vessels  containing  them,  wires  used  in  transferring  bacteria  from 
one  culture  medium  to  another,  instruments  used  in  making 
autopsies,  etc.,  must  be  sterilised.  These  objects  being  so 
different,  various  methods  are  necessary,  but  underlying  these 
methods  is  the  general  principle  that  all  bacteria  are  destroyed 
by  heat.  The  temperature  necessary  varies  with  different 
bacteria,  and  the  vehicle  of  heat  is  also  of  great  importance. 
The  two  vehicles  employed  are  hot  air  and  hot  water  or  steam. 
The  former  is  usually  referred  to  as  "  dry  heat,"  the  latter  as 
"moist  heat."  As  showing  the  different  effects  of  the  two 
vehicles,  Koch  found,  for  instance,  that  the  spores  of  bacillus 
anthracis,  which  were  killed  by  moist  heat  at  100°  C.,  in  one 
hour,  required  three  hours'  dry  heat  at  140°  C.  to  effect  death. 
Both  forms  of  heat  may  be  applied  at  different  temperatures — 
in  the  case  of  moist  heat  above  100°  C.,  a  pressure  higher  than 
that  of  the  atmosphere  must  of  course  be  present. 

A.  Sterilisation  by  Dry  Heat. 

A.  (1)  Red  Heat  or  Dull  Red  Heat.— Red  heat  is  used  for 
the  sterilisation  of  the  platinum  needles  which,  it  will  be  found, 
are  so  constantly  in  use.  A  dull  heat  is  used  for  cauteries,  the 
points  of  forceps,  and  may  be  used  for  the  incidental  sterilisation 
of  small  glass  objects  (cover-slips,  slides,  occasionally  when 
necessary  even  test-tubes),  care  of  course  being  taken  not  to 
melt  the  glass.  The  heat  is  obtained  by  an  ordinary  Bunsen 
burner. 

A.  (2)  Sterilisation  by  Dry  Heat  in  a  Hot- Air  Chamber.— 
The  chamber  (Fig.  2)  consists  of  an  outer  and  inner  case  of 
sheet  iron.  In  the  bottom  of  the  outer  there  is  a  large  hole. 
A  Bunsen  is  lit  beneath  this,  and  thus  plays  on  the  bottom  of 
the  inner  case,  round  all  of  the  sides  of  which  the  hot  air  rises 
and  escapes  through  holes  in  the  top  of  the  outer  case.  A 
thermometer  passes  down  into  the  interior  of  the  chamber,  half- 
way up  which  its  bulb  should  be  situated.  It  is  found  as  a 
matter  of  experience,  that  an  exposure  in  such  a  chamber  for 


STERILISATION   BY   MOIST   HEAT 


27 


one  hour  to  a  temperature  of  170°  C.,  is  sufficient  to  kill  all  the 
organisms  which  usually  pollute 
articles  in  a  bacteriological 
laboratory,  though  circum- 
stances might  arise  where  this 
would  be  insufficient.  This 
means  of  sterilisation  is  used 
for  the  glass  flasks,  test-tubes, 
plates,  Petri's  dishes,  the  use  of 
which  will  be  described.  Such 
pieces  of  apparatus  are  thus 
obtained  sterile  and  dry.  It  is 
advisable  to  put  glass  vessels 
into  the  chamber  before  heat- 
ing it,  and  to  allow  them  to 
stand  in  it  after  sterilisation 
till  the  temperature  falls.  Sud- 
den heating  or  cooling  is  apt 
to  cause  glass  to  crack.  The 
method  is  manifestly  unsuitable 
for  food  media. 


FIG.  2. — Hot-air  steriliser. 


B.  Sterilisation  by  Moist  Heat. 

B.  (1)  By  Boiling. — The  boiling  of  a  liquid  for  five  minutes 
is  sufficient  to  kill  ordinary  germs  if  no 
spores  be  present,  and  this  method  is  useful 
for  sterilising  distilled  or  tap  water  which 
may  be  required  in  various  manipulations. 
It  is  best  to  sterilise  knives  and  instruments 
used  in  autopsies  by  boiling  in  water  to 
which  a  little  sodium  carbonate  has  been 
added  to  prevent  rusting.  Twenty  minutes' 
boiling  will  here  be  sufficient.  The  boiling 
of  any  fluid  at  100°  C.  for  one  and  a  half 
hours  will  ensure  sterilisation  under  almost 
any  circumstances. 

B.  (2)  By  Steam  at  100°  C.— This  is  by 
far  the  most  useful  means  of  sterilisation. 
It  .may  be  accomplished  in  an  ordinary 
potato  steamer  placed  on  a  kitchen  pot. 
The  apparatus  ordinarily  used  is  "Koch's 

steam   steriliser"  (Fig.    3).      This  consists 

FIG.  3.— Koch's  steam  of   a  tal1  metal  cylinder  on  legs,  provided 
steriliser.  with    a    lid,    and    covered    externally    by 


28       METHODS    OF    CULTIVATION   OF   BACTERIA 

some  bad  conductor  of  heat,  such  as  felt  or  asbestos.  A 
perforated  tin  diaphragm  is  fitted  in  the  interior  at  a  little 
distance  above  the  bottom,  and  there  is  a  tap  at  the  bottom 
by  which  water  may  be  supplied  or  withdrawn.  If  water 
to  the  depth  of  3  inches  be  placed  in  the  interior  and  heat 
applied,  it  will  quickly  boil,  and  the  steam  streaming  up  will 
surround  any  flask  or  other  object  standing  on  the  diaphragm. 
Here  no  evaporation  takes  place  from  any  medium  as  it  is 
surrounded  during  sterilisation  by  an  atmosphere  saturated  with 
water  vapour.  It  is  convenient  to  have  the  cylinder  tall  enough 
to  hold  a  litre  flask  with  a  funnel  7  inches  in  diameter  standing 
in  its  neck.  The  funnel  may  be  supported  by  passing  its  tube 
through  a  second  perforated  diaphragm  placed  in  the  upper  part 
of  the  steam  chamber.  With  such  a  "  Koch  "  in  the  laboratory 
a  hot-water  filter  is  not  needed.  As  has  been  said,  one  and  a 
half  hour's  steaming  will  sterilise  any  medium,  but  in  the  case 
of  media  containing  gelatin  such  an  exposure  is  not  practic- 
able, as  with  long  boiling,  gelatin  tends  to  lose  its  physical 
property  of  solidification.  The  method  adopted  in  this  case 
is  to  steam  for  a  quarter  of  an  hour  on  each  of  three  succeeding 
days. 

This  is  a  modification  of  what  is  known  as  "  Tyndall's  intermittent 
sterilisation."  The  fundamental  principle  of  this  method  is  that  all 
bacteria  in  a  non-spored  form  are  killed  by  the  temperature  of  boiling 
water,  while  if  in  a  spored  form  they  may  not  be  thus  killed.  Thus  by 
the  sterilisation  on  the  first  day  all  the  nori-spored  forms  are  destroyed — 
the  spores  remaining  alive.  During  the  twenty-four  hours  which 
intervene  before  the  next  heating,  these  spores,  being  in  a  favourable 
medium,  are  likely  to  assume  the  non-spored  form.  The  next  heating 
kills  these.  In  case  any  may  still  not  have  changed  their  spored  form, 
the  process  is  repeated  on  a  third  day.  Experience  shows  that  usually 
the  medium  can  now  be  kept  indefinitely  in  a  sterile  condition. 

Steam  at  100°  C.  is  therefore  available  for  the  sterilisation  of 
all  ordinary  media.  In  using  the  Koch's  steriliser,  especially 
when  a  large  bulk  of  medium  is  to  be  sterilised,  it  is  best  to 
put  the  media  in  while  the  apparatus  is  cold,  in  order  to  make 
certain  that  the  whole  of  the  food  mass  reaches  the  temperature 
of  100°  C.  The  period  of  exposure  is  reckoned  from  the  time 
boiling  commences  in  the  water  in  the  steriliser.  At  any  rate 
allowance  must  always  be  made  for  the  time  required  to  raise 
the  temperature  of  the  medium  to  that  of  the  steam  surrounding  it. 

If  we  wish  to  use  such  a  substance  as  blood  serum  as  a 
medium,  the  albumin  would  be  coagulated  by  a  temperature  of 
100°  C.  Therefore  other  means  have  to  be  adopted  in  this  case. 


STERILISATION   BY   HIGH-PRESSURE   STEAM      29 


The  temperature 


oo 


oooo 


B.  (3)  Sterilisation  by  Steam  at  High  Pressure. — This  is 
the  most  rapid  and  effective  means  of  sterilisation.  It  is  effected 
in  an  autoclave  (Fig.  4).  This  is  a  gun-metal  cylinder  supported 
in  a  cylindrical  sheet-iron  case ;  its  top  is  fastened  down  with 
screws  and  nuts  and  is  furnished  with  a  safety  valve,  pressure- 
gauge,  and  a  hole  for  thermometer.  As  in  the  Koch's  steriliser, 
the  contents  are  supported  on  a  perforated  diaphragm.  The 
source  of  heat  is  a  large  Bunsen  beneath, 
employed  is  usually  115°  C.  or  120°  C. 
To  boil  at  115°  C.,  water  requires  a  pres- 
sure of  about  23  Ibs.  to  the  square  inch  (i.e. 
8  Ibs.  plus  the  15  Ibs.  of  ordinary  atmo- 
spheric pressure).  To  boil  at  120°  C.,  a 
pressure  of  about  30  Ibs.  (i.e.  15  Ibs.  plus 
the  usual  pressure)  is  necessary.  In  such  an 
apparatus  the  desired  temperature  is  main- 
tained by  adjusting  the  safety  valve  so  as 
to  blow  off  at  the  corresponding  pressure. 
One  exposure  of  media  to  such  temperatures 
for  a  quarter  of  an  hour  is  amply  sufficient 
to  kill  all  organisms  or  spores.  Here,  again, 
care  must  be  taken  when  gelatin  is  to  be 
sterilised.  It  must  not  be  exposed  to  a 
temperature  above  105°  C.,  and  is  best 
sterilised  by  the  intermittent  method.  Cer- 
tain precautions  are  necessary  in  using  the 
autoclave.  In  all  cases  it  is  necessary  to 
allow  the  apparatus  to  cool  well  below  1 00° 
C.,  before  opening  it  or  allowing  steam  to 
blow  off,  otherwise  there  will  be  a  sudden 
development  of  steam  when  the  pressure  is  removed,  and  fluid 
media  will  be  blown  out  of  the  flasks.  Sometimes  the  instrument 
is  not  fitted  with  a  thermometer.  In  this  case  care  must  be 
taken  to  expel  all  the  air  initially  present,  otherwise  a  mixture 
of  air  and  steam  being  present,  the  pressure  read  off  the  gauge 
cannot  be  accepted  as  an  indication  of  the  temperature.  Further, 
care  must  be  taken  to  ensure  the  presence  of  a  residuum  of 
water  when  steam  is  fully  up,  otherwise  the  steam  is  super- 
heated, and  the  pressure,  on  the  gauge  again  does  not  indicate 
the  temperature  correctly. 

B.  (4)  Sterilisation  at  Low  Temperatures. — Most  organisms 
in  a  non-spored  form  are  killed  by  a  prolonged  exposure  to  a 
temperature  of  57°  C.  This  fact  has  been  taken  advantage  of  for 
the  sterilisation  of  blood  serum,  which  will  coagulate  if  exposed  to  a 


FIG.  4. — Autoclave. 

a.  Safety-valve. 
&.  Blow-off  pipe, 
c.  Gauge. 


30      METHODS   OF   CULTIVATION   OF   BACTERIA 


FIG.  5. — Steriliser  for  blood 
serum. 


temperature  above  that  point.  Such  a 
medium  is  sterilised  on  Tyndall's  prin- 
ciple by  exposing  it  for  an  hour  at  57° 
C.  for  eight  consecutive  days,  it  being 
allowed  to  cool  in  the  interval  to  the 
room  temperature.  The  apparatus  shown 
in  Fig.  5  is  a  small  hot-water  jacket 
heated  by  a  Bunsen  placed  beneath 
it,  the  temperature  being  controlled 
by  a  gas  regulator.  To  ensure  that 
the  temperature  all  around  shall  be 
the  same,  the  lid  also  is  hollow  and 
filled  with  water,  and  there  is  a  special 
gas  burner  at  the  side  to  heat  it. 
This  is  the  form  originally  used,  but 
serum  sterilisers  are  now  constructed 
in  which  the  test-tubes  are  placed  in 
the  sloped  position,  and  in  which 
inspissation  (vide  p.  40)  can  after- 
wards be  performed  at  a  higher 
temperature. 


THE  PREPARATION  OF  CULTURE  MEDIA. 

The  general  principle  to  be  observed  in  the  artificial  culture 
of  bacteria  is  that  the  medium  used  should  approximate  as 
closely  as  possible  to  that  on  which  the  bacterium  grows  naturally. 
In  the  case  of  pathogenic  bacteria  the  medium  therefore  should 
resemble  the  juices  of  the  body.  The  serum  of  the  blood 
satisfies  this  condition  and  is  often  used,  but  its  application  is 
limited  by  the  difficulties  in  its  preparation  and  preservation. 
Other  media  have  been  found  which  can  support  the  life  of  all 
the  pathogenic  bacteria  isolated.  These  consist  of  proteids  or 
carbohydrates  in  a  fluid,  semi-solid,  or  solid  form,  in  a  trans- 
parent or  opaque  condition.  The  advantage  of  having  a  variety 
of  media  lies  in  the  fact  that  growth  characters  on  particular 
media,  non-growth  on  some  and  growth  on  others,  etc.,  constitute 
specific  differences  which  are  valuable  in  the  identification  of 
bacteria.  The  most  commonly  used  media  have  as  their  basis 
a  watery  extract  of  meat.  Most  bacteria  in  growing  in  such  an 
extract  cause  only  a  grey  turbidity.  A  great  advance  resulted 
when  Koch,  by  adding  to  it  gelatin,  provided  a  transparent  solid 
medium  in  which  growth  characteristics  of  particular  bacteria 


PREPARATION  OF  MEAT  EXTRACT 


31 


become  evident.  Many  organisms,  however,  grow  best  at  a 
temperature  at  which  this  nutrient  gelatin  is  fluid,  and  there- 
fore another  gelatinous  substance  called  agar,  which  does  not 
melt  below  98°  C.,  was  substituted.  Bouillon  made  from  meat 
extract,  gelatin,  and  agar  media,  and  the  modifications  of 
these,  constitute  the  chief '  materials  in  which  bacteria  are 
grown. 

Preparation  of  Meat  Extract. 

The  flesh  of  the  ox,  calf,  or  horse  is  usually  employed. 
Horse-flesh  has  the  advantage  of  being  cheaper  and  containing 
less  fat  than  the  others ;  though  generally  quite  suitable,  it  has 
the  disadvantage  for  certain  purposes  of  containing  a  larger 
proportion  of  fermentable  sugar.  The 
flesh  must  be  freed  from  fat,  and  finely 
minced.  To  a  pound  of  mince  add  1000 
c.c.  distilled  water,  and  mix  thoroughly 
in  a  shallow  dish.  Set  aside  in  a  cool 
place  for  twenty-four  hours.  Skim  off 
any  fat  present,  removing  the  last  traces 
by  stroking  the  surface  of  the  fluid  with 
pieces  of  filter  paper.  Place  a  clean  linen 
cloth  over  the  mouth  of  a  large  filter 
funnel,  .and  strain  the  fluid  through  it 
into  a  flask.  Pour  the  minced  meat  into 
the  cloth,  and  gathering  up  the  edges 
of  the  latter  in  the  left  hand,  squeeze 
out  the  juice  still  held  back  in  the  con- 
tained meat.  Finish  this  expression  by  putting  the  cloth  and  its 
contents  into  a  meat  press  (Fig.  6),  similar  to  that  used  by 
pharmacists  in  preparing  extracts ;  thus  squeeze  out  the  last  drops. 
The  resulting  sanguineous  fluid  contains  the  soluble  albumins  of 
the  meat,  the  soluble  salts,  extractives,  and  colouring  matter, 
chiefly  haemoglobin.  It  is  now  boiled  thoroughly  for  two  hours, 
by  which  process  the  albumins  coagulable  by  heat  are  coagulated. 
Strain  now  through  a  clean  cloth,  boil  for  another  half  hour,  and 
filter  through  white  Swedish  filter  paper  (best,  C.  Schleicher  u. 
Schull,  No.  595).  Make  up  to  1000  c.c.  with  distilled  water. 
The  resulting  fluid  ought  to  be  quite  transparent,  of  a  yellowish 
colour  without  any  red  tint.  If  there  is  any  redness,  the  fluid 
must  be  reboiled  and  filtered  till  this  colour  disappears,  other- 
wise in  the  later  stages  it  will  become  opalescent.  A  large 
quantity  of  the  extract  may  be  made  at  a  time,  and  what  is  not 


FIG.  6. — Meat  press. 


32      METHODS   OF   CULTIVATION   OF   BACTERIA 

immediately  required  is  put  into  a  large  flask,  the  neck  plugged 
with  cotton  wool,  and  the  whole  sterilised  by  methods  B 
(2)  or  (3).  This  extract  contains  very  little  albuminous  matter, 
and  consists  chiefly  of  the  soluble  salts  of  the  muscle,  certain 
extractives,  and  altered  colouring  matters,  along  with  any  slight 
traces  of  soluble  proteid  not  coagulated  by  heat.  It  is  of  acid 
reaction.  We  have  now  to  see  how,  by  the  addition  of  proteid 
and  other  matter,  it  may  be  transformed  into  proper  culture 
media. 

1.  Bouillon  Media. — These  consist  of  meat  extract  with  the 
addition  of  certain  substances  to  render  them  suitable  for  the 
growth  of  bacteria. 

(1)  (a).  Peptone  Broth  or  Bouillon. — This  has  the  com- 
position : — 

Meat  extract     ....     1000  c.c. 
Sodium  chloride        ...  5  grms. 

Peptone  albumin        .          .         .          10     „ 

Boil  till  the  ingredients  are  quite  dissolved,  and  neutralise 
with  a  saturated  solution  of  sodium  hydrate.  Add  the  latter 
drop  by  drop,  shaking  thoroughly  between  each  drop  and  testing 
the  reaction  by  means  of  litmus  paper.  Go  on  till  the  reaction 
is  slightly  but  distinctly  alkaline.  Neutralisation  must  be 
practised  with  great  care,  as  under  certain  circumstances, 
depending  on  the  relative  proportions  of  the  different  phosphates 
of  sodium  and  potassium,  what  is  known  as  the  amphoteric 
reaction  is  obtained,  i.e.  red  litmus  is  turned  blue,  and  blue  red, 
by  the  same  solution.  The  sodium  hydrate  must  be  added  till 
red  litmus  is  turned  slightly  but  distinctly  blue,  and  blue  litmus 
is  not  at  all  tinted  red.  After  alkalinisation,  allow  the  fluid  to 
become  cold,  filter  through  Swedish  filter  paper  into  flasks,  make 
up  to  original  volume  with  distilled  water,  plug  the  flasks  with 
cotton  wool,  and  sterilise  by  methods  B  (2)  or  (3),  (pp.  27,  29). 
This  method  of  neutralisation  is  to  be  recommended  for  all 
ordinary  work. 

In  this  medium  the  place  of  the  original  albumins  of  the  meat  is 
taken  by  peptone,  a  soluble  proteid  not  coagulated  by  heat.  Here  it 
may  be  remarked  that  the  commercial  peptone  albumin  is  not  pure 
peptone,  but  a  mixture  of  albumoses  (see  footnote,  p.  165)  with  a  variable 
amount  of  pure  peptone.  The  addition  of  the  sodium  chloride  is 
necessitated  by  the  fact  that  alkalinisation  precipitates  some  of  the 
phosphates  and  carbonates  present.  Experience  has  shown  that  sodium 
chloride  can  quite  well  be  substituted.  The  reason  for  the  alkalinisation 
is  that  it  is  found  that  most  bacteria  grow  best  on  a  medium  slightly 
alkaline  to  litmus.  Some,  e.g.  the  cholera  vibrio,  will  not  grow  at  all 
on  even  a  slightly  acid  medium. 


STANDARDISING   THE   REACTION   OF   MEDIA     33 

Standardisation  of  Reaction  of  Media. — While  the  above 
procedure  of  dealing  with  the  reaction  of  a  medium  is  sufficient 
for  ordinary  work,  it  has  been  thought  advisable  to  have  a  more 
exact  method  for  making  media  to  be  used  in  growing  organisms, 
the  growth  characteristics  of  which  are  to  be  described  for 
systematic  purposes.  Such  a  method  should  also  be  used  in 
studying  the  changes  in  reaction  produced  in  a  medium  by  the 
growth  of  bacteria.  It,  however,  involves  considerable  difficulty, 
and  should  not  be  undertaken  by  the  beginner.  It  entails  the 
preparation  of  solutions  of  acid  and  alkali  which  may  be  used 
for  determining  the  original  reaction  of  the  medium,  and  for 
accurately  making  it  of  a  definite  degree  of  alkalinity.  Normal l 
and  decinormal  solutions  of  sodium  hydrate  and  hydrochloric 
acid  are  used. 

Preparation  of  Standard  Solutions. — The  first  requisites  here  are 
normal  solutions  of  acid  and  alkali.  The  latter  is  prepared  as  follows  : 
— 85  grammes  of  pure  sodium  bicarbonate  are  heated  to  dull  redness 
for  ten  minutes  in  a  platinum  vessel  and  allowed  to  cool  in  an  exsiccator ; 
just  over  54  grammes  of  sodium  carbonate  should  now  be  present.  Any 
excess  is  quickly  removed,  and  the  rest  being  dissolved  in  one  litre  of 
distilled  water,  a  normal  solution  is  obtained.  A  measured  quantity  is 
placed  in  a  porcelain  dish,  and  a  few  drops  of  a  '5  per  cent  solution  of 
phenol-phthaleine  in  neutral  methylated  spirit  is  added  to  act  as  indicator. 
The  alkali  produces  in  the  latter  a  brilliant  rose  pink  which,  however, 
disappears  on  the  least  excess  of  acid  being  present.  The  mixture  is 
boiled  and  a  solution  of  hydrochloric  acid  of  unknown  strength  is  run 
into  the  dish  from  a  burette  till  the  colour  goes  and  does  not  return 
after  very  thorough  stirring.  The  strength  of  the  acid  can  then  be 
calculated,  and  a  normal  solution  can  be  obtained.  From  these  two 
solutions  any  strength  of  acid  or  alkali  (such  as  the  decinormal  solution 
of  NaOH  mentioned  below)  may  be  derived. 

As  Eyre  has  suggested,  the  reaction  of  a  medium  may  be 
conveniently  expressed  by  the  sign  +  or  -  to  indicate  acid  or 
alkaline  respectively,  and  a  number  to  indicate  the  number  of 
cubic  centimetres  of  normal  acid  or  alkaline  solution  necessary 
to  make  a  litre  of  the  medium  neutral  to  phenol-phthaleine. 
Thus,  for  example,  "reaction  =  -  15,"  will  mean  that  the  medium 
is  alkaline,  and  requires  15  c.c.  of  normal  HC1  to  make  a  litre 

1  A  "normal"  solution  of  any  salt  is  prepared  by  dissolving  an  "equivalent" 
weight  in  grammes  of  that  salt  in  a  litre  of  distilled  water.  If  the  metal  of 
the  salt  be  monovalent,  i.e.  if  it  be  replaceable  in  a  compound  by  one  atom 
of  hydrogen  (e.g.  sodium),  an  equivalent  is  the  molecular  weight  in 
grammes.  In  the  case  of  N'aCl,  it  would  be  58'5  grammes  (atomic  weight  of 
Na  =  23,  of  Cl  =  35'5).  If  the  metal  be  bivalent,  i.e.  requiring  two  atoms  of  H 
for  its  replacement  in  a  compound  (e.g.  calcium),  an  equivalent  is  the 
molecular  weight  in  grammes  divided  by  two.  Thus  in  the  case  of  CaCl2  an 
equivalent  would  be  55'5  grammes  (atomic  weight  of  Ca  =  40,  of  C12-71). 

3 


34     METHODS   OF   CULTIVATION   OF   BACTEEIA 

neutral.  It  has  been  found  that  when  a  medium  such  as  bouillon 
reacts  neutral  to  litmus,  its  reaction  to  phenol-phthaleine,  accord- 
ing to  the  above  standard,  is  on  the  average  +  25.  Now  as 
litmus  was  originally  introduced  by  Koch,  and  as  nearly  all 
bacterial  research  has  been  done  with  media  tested  by  litmus, 
it  is  evidently  difficult  to  say  exactly  what  precise  degree  of 
alkalinity  is  the  optimum  for  bacterial  growth.  It  is  probably 
safe  to  say,  however,  that  when  a  medium  has  been  rendered 
neutral  to  phenol-phthaleine  by  the  addition  of  NaOH,  the 
optimum  degree  is  generally  attained  by  the  addition  of  from 
10  to  15  c.c.  of  normal  HC1  per  litre,  i.e.  the  optimum  reaction 
is  + 10  to  + 15.  In  other  words,  the  optimum  reaction  for 
bacterial  growth  lies,  as  Fuller  has  pointed  out,  about  midway 
between  the  neutral  point  indicated  by  phenol-phthaleine  and 
the  neutral  point  indicated  by  litmus. 

The  only  objection  to  the  use  of  phenol-phthaleine  is  that  its 
action  is  somewhat  vitiated  if  free  CO2  be  present.  This  can 
be  completely  obviated  as  follows.  Before  testing  any  medium 
it  is  boiled  in  the  porcelain  dish  into  which  titration  takes  place. 
The  soda  solutions  are  best  stored  in  bottles  such  as  that  shown 
in  Fig.  44,  having  on  the  air  inlet  a  little  bottle  filled  with  soda 
lime  with  tubes  fitted  as  in  the  large  one.  The  C02  of  the  air 
which  passes  through  is  thus  removed. 

Method. — The  following  procedure  includes  most  of  the 
improvements  introduced  by  Eyre.  The  medium  with  all  its 
constituents  dissolved  is  filtered  and  then  heated  for  about  45 
minutes  in  the  steamer,  the  maximum  acidity  being  reached 
after  this  time.  Of  the  warm  medium  take  25  c.c.  and  put  in 
a  porcelain  dish,  add  25  c.c.  distilled  water,  and  1  c.c.  phenol- 
phthaleine  solution.  Run  in  decinormal  soda  till  neutral  point 
is  reached,  indicated  by  the  first  trace  of  pink  colour,  the  mixture 
being  kept  hot.1  Repeat  process  thrice,  and  take  the  mean ; 
this  divided  by  10  will  give  the  amount  (x)  of  normal  soda 
required  to  neutralise  25  c.c.  of  medium ;  then  40  x  =  amount 
necessary  to  neutralise  a  litre;  and  40  #-10  =  amount  of 
normal  soda  necessary  to  give  a  litre  its  optimum  reaction. 
Then  measure  the  amount  of  medium  to  be  dealt  with,  and  add 
the  requisite  amount  of  soda  solution. 

1  The  beginner  may  find  considerable  difficulty  in  recognising  the  first 
tint  of  pink  in  the  yellow  bouillon. .  A  good  way  of  getting  over  this  is  to 
take  two  samples  of  the  medium,  adding  the  indicator  to  one  only  ;  then  to 
run  the  soda  into  these  from  separate  burettes  ;  for  each  few  drops  run  into 
the  medium  containing  the  indicator  the  same  amount  is  run  into  the  other. 
Thus  the  recognition  of  the  first  permanent  change  in  tint  will  be  at  once 
recognised  by  comparing  the  two  lots  of  solution. 


GELATIN   MEDIA 


35 


Eyre  uses  a  soda  solution  of  ten  times  normal  strength,  which 
is  delivered  out  of  a  1  c.c.  pipette  divided  into  hundredths ;  this 
obviates,  to  a  large  extent,  the  error  introduced  by  increasing 
the  bulk  of  the  medium  on  the  addition  of  the  neutralising 
solution. 

1  (b).  Glucose  Broth. — To  the  other  constituents  of  1  (a) 
there  is  added  1  or  2  per  cent  of  grape  sugar.  The  steps  in  the 
preparation  are  the  same.  Glucose  being  a  reducing  agent, 
no  free  oxygen  can  exist  in  a  medium  containing  it,  and  there- 
fore glucose  broth  is  used  as  a  culture  fluid  for  anaerobic 
organisms. 

1  (c).  Glycerin  Broth. — The  initial  steps  are  the  same  as  in 
1  (a),  but  after  filtration  6  to  8  per  cent  of  glycerin  (sp.  grav.  1'25) 
is  added.     This  medium    is   especially   used   for   growing   the 
tubercle  bacillus  when  the  soluble  products  of  the  growth  of  the 
latter  are  required. 

2.  Gelatin  Media. — These  are  simply  the  above  broths,  with 
gelatin  added  as  a  solidifying  body. 

2  (a).  Peptone  Gelatin  : — 


Meat  extract 
Sodium  chloride 
Peptone  albumin 
Gelatin 


.     1000  c.c. 

5  grms. 
10     „ 
100-150 


(the  "  gold  label "  gelatin  of  Coignet  et  Cie,  Paris,  is  the  best). 
The  gelatin  is  cut  into  small  pieces,  and  added  with  the  other 
constituents  to  the  extract ;  they  are 
then  thoroughly  melted  on  a  sand  bath, 
or  in  the  "  Koch."  The  fluid  medium 
is  then  rendered  slightly  alkaline,  as 
in  1  (a),  and  filtered  through  filter 
paper.  As  the  medium  must  not  be 
allowed  to  solidify  during  the  process, 
it  must  be  kept  warm.  This  is  effected 
by  putting  the  flask  and  funnel  into  a 
tall  Koch's  steriliser,  in  which  case 
the  funnel  must  be  supported  on  a 
tripod  or  diaphragm,  as  there  is  great 
danger  of  the  neck  of  the  flask  breaking 
if  it  has  to  support  the  funnel  and 
its  contents.  The  filtration  may  also 
be  carried  out  in  a  funnel  with  water- 
jacket  which  is  heated,  as  shown  in  Fig.  7.  Whichever  instrument 
be  used,  before  filtering  shake  up  the  melted  medium,  as  it  is  apt 


FIG.  7. — Hot- water  funnel. 


36     METHODS   OF   CULTIVATION   OF   BACTERIA 

while  melting  to  have  settled  into  layers  of  different  density. 
Sometimes  what  first  comes  through  is  turbid.  If  so,  replace  it 
in  the  unfiltered  part  :  often  the  subsequent  filtrate  in  such  cir- 
cumstances is  quite  clear.  A  litre  flask  of  the  finished  product 
ought  to  be  quite  transparent.  If,  however,  it  is  partially  opaque, 
add  the  white  of  an  egg,  shake  up  well,  and  boil  thoroughly  over 
the  sand  bath.  The  consequent  coagulation  of  the  albumin  carries 
down  the  opalescent  material,  and  on  making  up  with  distilled 
water  to  the  original  quantity  and  refiltering,  it  will  be  found  to 
be  clear.  The  flask  containing  it  is  then  plugged  with  cotton 
wool  and  sterilised,  best  by  method  B  (2),  p.  27.  If  the 
autoclave  be  used  the  temperature  employed  must  not  be  above 
105°  C.,  and  exposure  not  more  than  a  quarter  of  an  hour  on 
three  successive  days.  Too  much  boiling,  or  boiling  at  too  high 
a  temperature,  as  has  been  said,  causes  a  gelatin  medium  to 
lose  its  property  of  solidification.  The  exact  percentage  of 
gelatin  used  in  its  preparation  depends  on  the  temperature  at 
which  growth  is  to  take  place.  Its  firmness  is  its  most  valuable 
characteristic,  and  to  maintain  this  in  summer  weather,  15  parts 
per  100  are  necessary.  A  limit  is  placed  on  higher  percentages 
by  the  fact  that,  if  the  gelatin  be  too  stiff,  it  will  split  on  the 
perforation  of  its  substance  by  the  platinum  needle  used  in 
inoculating  it  with  a  bacterial  growth ;  1 5  per  cent  gelatin  melts 
at  about  24°  C. 

2  (6).  Glucose  Gelatin. — The  constituents  are  the  same  as 
2  (a),  with  the  addition  of  1  to  2  per  cent  of  grape  sugar.     The 
method  of  preparation  is  identical.     This  medium  is  used  for 
growing  anaerobic  organisms  at  the  ordinary  temperatures. 

3.  Agar  Media  (French,  "gelose  ").— The  disadvantage  of 
gelatin  is  that  at  the  blood  temperature  (38°  C.),  at  which  most 
pathogenic  organisms  grow  best,  it  is  liquid.  To  get  a  medium 
which  will  be  solid  at  this  temperature,  agar  is  used  as  the 
stiffening  agent  instead  of  gelatin.  Unlike  the  latter,  which  is 
a  proteid,  agar  is  a  carbohydrate.  It  is  derived  from  the  stems  of 
various  sea-weeds  growing  in  the  Chinese  seas,  popularly  classed 
together  as  "  Ceylon  Moss."  For  bacteriological  purposes  the  dried 
stems  of  the  seaweed  may  be  used,  but  there  is  in  the  market  a 
purified  product  in  the  form  of  a  powder ;  this  is  preferable. 

3  (a).   "  Ordinary  "  Agar. — This  has  the  following  composi- 
tion : — 

Meat  extract 1000  c.c. 

Sodium  chloride         .          .          .          .  5  grms. 

Peptone  albumin        .         .          .          .          10    „ 
Agar         .         .         .         .         .         .         15    „ 


AGAR   MEDIA  37 

Cut  up  the  agar  into  very  fine  fragments  (in  fact  till  it  is  as 
nearly  as  possible  dust),  add  to  the  meat  extract  with  the  other 
ingredients,  and  preferably  allow  to  stand  all  night.  Then  boil 
gently  in  a  water  bath  for  two  or  three  hours,  till  the  agar  is 
thoroughly  melted.  The  process  of  melting  may  be  hastened 
by  boiling  the  medium  in  a  sand  bath  and  passing  through  it  a 
stream  of  steam  generated  in  another  flask ;  the  steam  is  led 
from  the  second  flask  by  a  bent  glass  tube  passing  from  just 
beneath  the  cork  to  beneath  the  surface  of  the  medium  (Eyre). 
After  melting  render  slightly  alkaline  with  sodium  hydrate 
solution,  make  up  to  original  volume  with  distilled  water,  and 
filter.  Filtration  here  is  a  very  slow  process  and  is  best  carried 
out  in  a  tall  Koch's  steriliser.  In  doing  this,  it  is  well  to  put  a 
glass  plate  over  the  filter  funnel  to  prevent  condensation  water 
from  dropping  off  the  roof  of  the  steriliser  into  the  medium.  If 
a  slight  degree  of  turbidity  may  be  tolerated,  it  is  sufficient  to 
filter  through  a  felt  bag  or  jelly  strainer.  Plug  the  flask  con- 
taining the  filtrate,  and  sterilise  either  in  autoclave  for  fifteen 
minutes  or  in  Koch's  steriliser  for  one  and  a  half  hours.  Agar 
melts  just  below  100°  C.,  and  on  cooling  solidifies  about  39°  C. 

3  (6).  Glycerin  Agar. — To  3  (a)  after  filtration  add  6  to  8 
per  cent  of  glycerin  and  sterilise  as  above.  This  is  used 
especially  for  growing  the  tubercle  bacillus. 

3  (c).  Glucose  Agar. — Prepare  as  in  3  (a\  but  add  1  to  2 
per  cent  of  grape  sugar  along  with  agar.  This  medium  is  used 
for  the  culture  of  anaerobic  organisms  at  temperatures  above  the 
melting-point  of  gelatin.  It  is  also  a  superior  culture  medium 
for  some  aerobes,  e.g.  the  b.  diphtherise. 

These  bouillon,  gelatin,  and  agar  preparations  constitute 
the  most  frequently  used  media.  Growths  in  bouillon  do  not 
usually  show  any  characteristic  appearances  which  facilitate 
classification,  but-  such  a  medium  is  of  great  use  in  investigating 
the  soluble  toxic  products  of  bacteria.  The  most  characteristic 
developments  of  organisms  take  place  on  the  gelatin  media. 
These  have,  however,  the  disadvantage  of  not  being  available 
when  growth  is  to  take  place  at  any  temperature  above  24°  C. 
For  higher  temperatures  agar  must  be  employed.  Agar  is,  how- 
ever, never  so  transparent.  Though  quite  clear  when  fluid,  on 
solidifying  it  always  becomes  slightly  opaque.  Further,  growths 
upon  it  are  never  so  characteristic  as  those  on  gelatin.  It  is, 
for  instance,  never  liquefied,  whereas  some  organisms,  by  their 
growth,  liquefy  gelatin  and  others  do  not — a  fact  of  prime 
importance. 

Litmus  Media. — To  any  of  the  above  media  litmus  (French, 


38     METHODS   OF   CULTIVATION   OF   BACTERIA 

tournesol)  may  be  added  to  show  change  in  reaction  during 
bacterial  growth.  The  litmus  is  added,  before  sterilisation,  as 
a  strong  watery  solution  (e.g.  the  Kubel-Tiemann  solution,  vide 
p.  42)  in  sufficient  quantity  to  give  the  medium  a  distinctly 
bluish  tint.  During  the  development  of  an  acid  reaction  the 
colour  changes  to  a  pink  and  may  subsequently  be  discharged. 

Use  of  neutral  red. — This  dye  has  been  introduced  as  an  aid 
in  determining  the  presence  or  absence  of  members  of  the  b.  coli 
group,  especially  in  the  examination  of  water.  The  media  found 
most  suitable  are  agar  or  bouillon  containing  *5  per  cent  of 
glucose,  to  which  *5  per  cent  of  a  one  per  cent  watery  solution 
of  neutral  red  is  added.  The  use  of  these  media  and  their 
probable  value  are  described  below  (vide  Typhoid  Fever). 

Blood  Agar  :  Serum  Agar. — The  former  medium  was  intro- 
duced by  Pfeiffer  for  growing  the  influenza  bacillus,  and  it 
has  been  used  for  the  organisms  which  are  not  easily  grown  on 
the  ordinary  media,  e.g.  the  gonococcus  and  the  pneumococcus. 
Human  blood  or  the  blood  of  animals  may  be  used.  "  Sloped 
tubes  "  (vide  p.  48)  of  agar  are  employed  (glycerin  agar  is  not  so 
suitable).  Purify  a  finger  first  with  1-1000  corrosive  sublimate, 
dry,  and  then  wash  with  absolute  alcohol  to  remove  the  sublimate. 
Allow  the  alcohol  to  evaporate.  Prick  with  a  needle  sterilised 
by  heat,  and,  catching  a  drop  of  blood  in  the  loop  of  a  sterile 
platinum  wire  (vide  p.  49),  smear  it  on  the  surface  of  the  agar. 
The  excess  of  the  blood  runs  down  and  leaves  a  film  on  the 
surface.  Cover  the  tubes  with  india-rubber  caps,  and  incubate 
them  for  one  or  two  days  at  38°  C.  before  use,  to  make  certain 
that  they  are  sterile.  Agar  poured  out  in  a  thin  layer  in  a 
Petri  dish  may  be  smeared  with  blood  in  the  same  way  and 
used  for  cultures.  A  medium  composed  of  one  part  of  fresh 
blood  (drawn  aseptically)  and  two  parts  of  fluid  agar  at  40°  C., 
has  been  used  for  the  cultivation  of  the  bacillus  of  soft  sore. 

Serum  agar  is  prepared  in  a  similar  way  by  smearing  the 
surface  of  the  agar  with  blood  serum,  or  by  adding  a  few  drops 
of  serum  to  the  tube  and  then  allowing  it  to  flow  over  the 
surface. 

Peptone  Solution. 

A  simple  solution  of  peptone  (Witte)  constitutes  a  suitable 
culture  medium  for  many  bacteria.  The  peptone  in  the  propor- 
tion of  1  to  2  per  cent,  along  with  *5  per  cent  NaCl,  is  dissolved 
in  distilled  water  by  heating.  The  fluid  is  then  filtered,  placed 
in  tubes  and  sterilised.  The  reaction  is  usually  distinctly 
alkaline,  which  condition  is  suitable  for  most  purposes.  For 


BLOOD   SEEUM  39 

special  purposes  the  reaction  may  be  standardised.  In  such  a 
solution  the  cholera  vibrio  grows  with  remarkable  rapidity.  It 
is  also  much  used  for  testing  the  formation  of  indol  by  a 
particular  bacterium ;  and  by  the  addition  of  one  of  the  sugars 
to  it  the  fermentative  powers  of  an  organism  may  be  tested 
(p.  75).  Litmus  may  be  added  to  show  any  change  in  reaction. 

Blood  Serum. 

Koch  introduced  this  medium,  and  it  is  prepared  as  follows  : 
Plug  the  mouth  of  a  tall  cylindrical  glass  vessel  (say  of  1000  c.c. 
capacity)  with  cotton  wool,  and  sterilise  by  steaming  it  in  a 
Koch's  steriliser  for  one  and  a  half  hours.  Take  it  to  the  place 
where  a  horse,  ox,  or  sheep  is  to  be  killed.  When  the  artery 
or  vein  of  the  animal  is  opened,  allow  the  first  blood  which 
flows,  and  which  may  be  contaminated  from  the  hair,  etc.,  to 
escape  ;  fill  the  vessel  with  the  blood  subsequently  shed.  Carry 
carefully  back  to  the  laboratory  without  shaking,  and  place  for 
twenty-four  hours  in  a  cool  place,  preferably  an  ice  chest.  The 
clear  serum  will  separate  from  the  clotted  blood.  If  a  centrifuge 
is  available,  a  large  yield  of  serum  may  be  obtained  by  centri- 
fugalising  the  freshly  drawn  blood.  If  coagulation  has  occurred, 
the  clot  must  first  be  thoroughly  broken  up.  With  a  sterile  10 
c.c.  pipette,  transfer  this  quantity  of  serum  to  each  of  a  series 
of  test-tubes  which  must  previously  have  been  sterilised  by  dry 
heat.  The  serum  may,  with  all  precautions,  have  been  con- 
taminated during  the  manipulations,  and  must  be  sterilised. 
As  it  will  coagulate  if  heated  above  68°  C.,  advantage  must  be 
taken  of  the  intermittent  process  of  sterilisation  at  57°  C. 
[method  B  (4)].  It  is  therefore  kept  for  one  hour  at  this 
temperature  on  each  of  eight  successive  days.  It  is  always 
well  to  incubate  it  for  a  day  at  37°  C.  before  use,  to  see  that 
the  result  is  successful.  After  sterilisation  it  is  "inspissated," 
by  which  process  a  clear  solid  medium  is  obtained.  "Inspissa- 
tion  "  is  probably  an  initial  stage  of  coagulation,  and  is  effected 
by  keeping  the  serum  at  65°  C.  till  it  stiffens.  This  temperature 
is  just  below  the  coagulation  point  of  the  serum.  The  more 
slowly  the  operation  is  performed  the  clearer  will  be  the  serum. 
The  apparatus  used  for  the  purpose  is  one  of  the  various  forms 
of  serum  steriliser  (e.g.  Fig.  8),  generally  a  chamber  with  water- 
jacket  heated  with  a  Bunsen  below.  The  temperature  is  con- 
trolled by  a  gas  regulator,  and  such  an  apparatus  can,  by  altering 
the  temperature,  be  used  either  for  sterilisation  or  inspissation. 
As  is  evident,  the  preparation  of  this  medium  is  tedious,  but  its 


40     METHODS   OF   CULTIVATION  OF   BACTEEIA 


use  is  necessary  for  the  observation  of  particular  characteristics 
in  several  pathogenic  bacteria,  notably  the  tubercle  bacillus. 
Pleuritic  and  other  effusions  may  be  prepared  in  the  same  way, 
and  used  as  media,  but  care  must  be  taken  in  their  use,  as  we 
have  no  right  to  say  that  pathological  effusions  have  the  same 

chemical    composition    as 
normal  serum. 

If  blood  be  collected 
with  strict  aseptic  precau- 
tions, then  sterilisation  of 
the  serum  is  unnecessary. 
To  this  end  the  mouth  of 
the  cylinder  used  for  col- 
lecting the  blood,  instead 
of  being  plugged  with  wool, 
has  an  india-rubber  bung 
inserted  in  it  through 
which  two  bent  glass  tubes 
pass.  The  outer  end  of 
one  of  these  is  of  conveni- 
ent length,  and,  before 
sterilisation,  a  large  cap  of 
cotton  wool  is  tied  over  it ; 
the  other  tube  is  plugged 
with  a  piece  of  cotton  wool. 
In  the  slaughter-house  the 
cap  is  removed  and  the 
tube  is  inserted  into  the 
blood-vessel  as  a  cannula. 
The  cylinder  is  thus  easily 
filled.  Another  method  is 
to  conduct  the  blood  to 
the  cylinder  by  means  of 
a  sterilised  cannula  and 
india-rubber  tube,  the 
former  being  inserted  in 
the  blood-vessel.  In  every  case  the  serum  must  be  incubated 
before  use,  to  make  sure  that  it  is  sterile. 

Coagulated  Blood  Serum. — If  fresh  serum  be  placed  in 
sterile  tubes  and  be  steamed  in  the  sloped  position  for  an  hour 
it  coagulates,  and  there  is  thus  obtained  a  solid  medium  very 
useful  for  the  growth  of  the  diphtheria  bacillus  for  diagnostic 
purposes. 

Loffler's  Blood  Serum. — This  is   the  best  medium  for  the 


FIG.  8. — Blood  serum  inspissator. 


BLOOD   SERUM  41 

growth  of  the  b.  diphtheriae  and  may  be  used  for  other 
organisms.  It  has  the  following  composition.  Three  parts  of 
calf's  or  lamb's  blood  serum  are  mixed  with  one  part  ordinary 
neutral  peptone  bouillon  made  from  veal  with  1  per  cent  of  grape 
sugar  added  to  it.  Though  this  is  the  original  formula  it  can 
be  made  from  ox  or  sheep  serum  and  beef  bouillon  without  its 
qualities  being  markedly  impaired.  Sterilise  by  method  B  (4) 
as  above  (p.  29). 

Alkaline  Blood  Serum  (Lorrain  Smith's  Method). — To  each 
100  c.c.  of  the  serum  obtained  as  before,  add  1  to  1*5  c.c.  of  a 
10  per  cent  solution  of  sodium  hydrate  and  shake  gently.  Put 
sufficient  of  the  mixture  into  each  of  a  series  of  test-tubes,  and 
laying  them  on  their  sides,  sterilise  by  method  B  (2).  If  the 
process  of  sterilisation  be  carried  out  too  quickly,  bubbles  of  gas 
are  apt  to  form  before  the  serum  is  solid,  and  these  interfere  with 
the  usefulness  of  the  medium.  Dr.  Lorrain  Smith  informs  us 
that  this  can  be  obviated  if  the  serum  be  solidified  high  up  in  the 
Koch's  steriliser,  in  which  the  water  is  allowed  only  to  simmer. 
In  this  case  sterilisation  ought  to  go  on  for  one  and  a  half  hours. 
A  clear  solid  medium  (consisting  practically  of  alkali-albumin)  is 
thus  obtained,  and  he  has  found  it  of  value  for  the  growth  of  the 
organisms  for  which  Koch's  serum  is  used,  and  especially  for  the 
growth  of  the  b.  diphtheriae.  Its  great  advantage  is  that  aseptic 
precautions  in  obtaining  blood  from  the  animal  are  not  necessary, 
and  it  is  easily  sterilised. 

Marmorek's  Serum  Media. — There  has  always  been  a  diffi- 
culty in  maintaining  the  virulence  of  cultures  of  the  pyogenic 
streptococci,  but  Marmorek  has  succeeded  in  doing  so  by  growing 
them  on  the  following  media,  which  are  arranged  in  the  order  of 
their  utility  : — 

1.  Human  serum  2  parts,  bouillon  1  part. 

2.  Pleuritic  or  ascitic  serum  1  part,  bouillon  2  parts. 

3.  Asses'  or  mules'  serum  2  parts,  bouillon  1  part. 

4.  Horse  serum  2  parts,  bouillon  1  part. 

Human  serum  can  be  obtained  from  the  blood  shed  in 
venesection,  the  usual  aseptic  precautions  being  taken.  In  the 
case  of  these  media,  sterilisation  is  effected  by  method  B  (4),  and 
they  are  used  fluid. 

Hiss's  Serum  Water  Media. — These  are  composed  of  one 
part  of  ox's  serum  and  three  parts  of  distilled  water  with  1 
per  cent  litmus  ;  various  sugars  in  a  pure  condition  are  added  in 
the  proportion  of  1  per  cent.  The  development  of  acid  by 
fermentation  is  shown  by  the  alteration  of  the  colour  and  by 


42     METHODS   OF   CULTIVATION   OF   BACTERIA 

coagulation  of  the  medium.  These  media  do  not  coagulate  at 
100°  C.  and  thus  can  be  sterilised  in  the  steam  steriliser. 
They  have  been  extensively  used  by  American  workers  in 
studying  the  fermentative  properties  of  the  b.  dysenteriae, 
b.  coli,  etc. 

Drigalski  and  Conradi's  Medium. — This  is  one  of  the  media  used  for 
the  study  of  intestinal  bacteria  and  especially  for  the  isolation  of  the 
typhoid  group  of  organisms,  (a)  Three  pounds  of  meat  are  treated  with 
two  litres  of  water  overnight  ;  the  fluid  is  separated  as  usual,  boiled 
for  an  hour,  filtered,  and  there  are  added  20  grammes  Witte's  peptone, 
20  grammes  nutrose,1  10  grammes  sodium  chloride  ;  the  mixture  is  then 
boiled  for  an  hour,  60  grammes  finest  agar  are  added,  and  it  is  placed  in 
the  autoclave  till  melted  (usually  one  hour)  ;  it  is  then  rendered  slightly 
alkaline  to  litmus,  filtered,  and  boiled  for  half  an  hour.  (6)  260  c.c.  Kubel- 
Tiemann  litmus 2  solution  is  boiled  for  ten  minutes,  30  grammes  milk 
sugar  (chemically  pure)  are  added,  and  the  mixture  is  boiled  for  fifteen 
minutes  ;  (a)  and  (b)  are  then  mixed  hot,  well  shaken,  and,  if  necessary, 
the  slightly  alkaline  reaction  restored.  There  are  then  added  4  c.c.  of 
a  10  per  cent  sterile  solution  of  water-free  sodium  hydrate  and  20  c.c.  of 
a  freshly  prepared  solution  made  by  dissolving  '1  gramme  crystal- violet 
B,  Hoechst,  in  100  c.c.  hot  sterile  distilled  water.  This  is  the  finished 
medium,  and  great  care  must  be  taken  not  to  overheat  it  or  to  heat  it  too 
long,  as  changes  in  the  lactose  may  be  originated.  It  is  convenient  to 
distribute  the  medium  in  80  c.c.  flasks. 

The  principle  of  the  medium  is  that  while  there  is  a  food  supply  very 
favourable  to  the  b.  typhosus  and  the  b.  coli  the  antiseptic  action  of  the 
crystal-violet  tends  to  inhibit  the  growth  of  other  bacteria  likely  to 
occur  in  material  which  has  been  subjected  to  intestinal  contamination. 
In  examining  faeces  a  little  is  rubbed  up  in  from  ten  to  twenty  times  its 
volume  of  sterile  normal  salt  solution  ;  in  the  case  of  urine  or  water  the 
fluid  is  centrifugalised  and  the  deposit  or  lower  portion  is  used  for  the 
inoculation  procedures. 

For  use  the  medium  is  distributed  in  Petri  capsules  in  a  rather  thicker 
layer  than  is  customary  in  an  ordinary  plate.  The  sheet  of  medium 
must  be  transparent,  but  must  not  be  less  than  2  mm.  in  thickness— 
in  fact,  ought  to  be  about  4  mm.  After  being  poured,  the  capsules  are 
left  with  the  covers  off  for  an  hour  or  so,  to  allow  the  superficial 
layers  of  the  medium  to  become  set  hard.  The  effect  of  this  is 
that  during  incubation  no  water  of  condensation  forms  on  the  lid 
of  the  capsule,  and  thus  the  danger  of  this  fluid  dropping  on  to  the 
developing  colonies  is  avoided.  The  antiseptic  nature  of  the  crystal- 
violet  is  sufficient  to  prevent  the  growth  of  any  aerial  organisms  falling 

1  Nutrose  is  an  alkaline  preparation  of  casein. 

2  The  litmus  solution   is  made  as  follows.     Solid  commercial  litmus  is 
digested  with  pure  spirit  at  30°  C.  till  on  adding  fresh  alcohol  the  latter 
becomes  only  of  a  light  violet  colour.     A  saturated  solution  of  the  residue  is 
then  made  in  distilled  water  and  filtered.     When  this  is  diluted  with  a  little 
distilled  water  it  is  of  a  violet  colour,  which  further  dilution  turns  to  a  pure 
blue.     To  such  a  blue  solution  very  weak  sulphuric  acid  (made  by  adding 
two  drops  of  dilute  sulphuric  acid  to  200  c.c.  water)  is  added  till  the  blue 
colour  is  turned  to  a  wine-red.    Then  the  saturated  solution  of  the  dye  is  added 
till  the  blue  colour  returns. 


MACCONKEY'S   BILE-SALT   MEDIA  43 

on  the  agar  during  its  exposure  to  the  air.  The  plates  are  usually 
inoculated  by  means  of  a  glass  spatula  made  by  bending  three  inches  of  a 
piece  of  glass  rod  at  right  angles  to  the  rest  of  the  rod.  This  part  is 
dipped  in  the  infective  material  and  smeared  in  all  directions  over  the 
surfaces  of  three  or  four  plates  successively  without  any  intervening 
sterilisation.  The  plates  are  again  exposed  to  the  air  after  inoculation 
for  half  an  hour  and  then  incubated  for  twenty-four  hours.  At  the  end 
of  such  a  period  b.  coli  colonies  are  2  to  6  mm.  in  diameter,  stained 
distinctly  red,  and  are  non-transparent.  Colonies  of  the  b.  typhosus  are 
seldom  larger  than  2  mm.,  they  are  blue  or  bluish- violet  in  colour,  are 
glassy  and  dew-like  in  character,  and  have  a  single  contour.  Sometimes 
in  the  plates  b.  subtilis  and  its  congeners  appear,  and  colonies  of  these 
organisms  have  a  blue  colour.  Their  growth  is,  however,  more  exuberant 
than  that  of  the  typhoid  bacillus, — being  often  heaped  up  in  the  centre, 
— and  the  contour  of  the  colony  is  often  double. 

MacConkey's  Bile-Salt  Media. — These  media  were  introduced  for  the 
purpose  of  differentiating  the  intestinal  bacteria,  and  have  been  exten- 
sively used  for  the  study  of  the  b.  coli,  b.  typhosus,  b.  dysenteries,  etc. 
The  characteristic  ingredients  are  bile  salts  and  various  sugars.  The 
stock  solution  is  the  following: — Commercial  sodium  taurocholate,  0*5 
gramme  ;  Witte's  peptone,  2'0  grammes  ;  distilled  water,  100  c.c. 
For  a  liquid  medium  there  is  added  to  this  '5  per  cent  of  a  freshly 
prepared  1  per  cent  solution  of  neutral  red1  and  the  sugar, — when 
glucose  is  used  0'5  per  cent  is  added,  in  the  case  of  other  sugars  1 
per  cent.  The  fluid  is  distributed  in  Durham's  fermentation  tubes  and 
sterilised  in  the  steamer  for  ten  minutes  on  two  successive  days,  care 
being  taken  not  to  overheat  the  medium. 

For  bile-salt  agar  1'5  to  2  per  cent  agar  is  dissolved  in  the  stock 
solution  in  the  autoclave,  if  necessary  cleared  with  white  of  egg  and 
filtered.  Neutral  red  and  a  sugar  are  added,  as  in  the  case  of  the  liquid 
medium.  As  with  Drigalski's  medium,  it  is  well  to  sterilise  it  in  flasks 
containing  80  c.c.,  this  being  an  amount  sufficient  for  three  Petri 
capsules.  When  this  medium  is  used  for  examining  urine  or  faeces, 
plates  are  inoculated  as  with  Drigalski's  medium  (supra}  ;  for  its  use  in 
water  examinations  see  p.  136. 

In  the  bile-salt  bouillon  the  formation  of  both  acid  and  gas  is 
observed  if  such  formation  occurs,  and  in  the  bile-salt  agar  acid  produc- 
tion is  recognised  by  the  red  colour  of  the  colonies  of  the  acid-producing 
organisms. 

MacConkey's  original  medium  was  a  1  per  cent  bile-salt  lactose  agar 
with  no  indicator,  and  was  used  for  the  detection  of  intestinal  bacteria 
in  water.  Such  a  medium  is  unfavourable  to  all  the  common  spore- 
bearing  organisms  found  in  water,  and  by  incubating  at  42°  C.  tubes,  in 
which  there  is  probably  a  mixed  infection  from  such  a  source,  the  growth 
of  most  other  water  bacteria  is  inhibited.  B.  coli  and  b.  typhosus,  on 
the  other  hand,  grow  readily.  With  the  former  the  surface  colonies  are 
broad,  irregular,  and  flat,  of  opaque  colour,  and  with  a  small  spot  of 
yellow  or  orange  in  the  centre,  and  the  colony  is  surrounded  by  a  haze  ; 
the  deep  colonies  are  lens-shaped,  of  orange  colour,  and  are  likewise 
surrounded  by  a  haze.  With  the  typhoid  organism  at  the  end  of  forty-eight 
hours  the  surface  colonies  are  small,  round,  raised,  and  semi-transparent, 
while  the  deep  colonies  are  lens-shaped,  white,  and  have  no  surrounding 

1  Neutral  red  gives  a  deep  crimson  with  acids  and  a  yellow -red  with 
alkalies. 


44     METHODS   OF   CULTIVATION   OF   BACTERIA 

haze.  The  haze  in  the  case  of  b.  coli  is  due  to  the  ready  production  of 
acid  from  the  lactose  causing  a  precipitate  of  the  taurocholate.  Any  other 
organism  capable  of  producing  acid  from  lactose  will  give  a  similar 
reaction,  and  the  haze  can  be  readily  cleared  up  by  floating  a  drop  of 
ammonia  on  the  surface  of  the  medium.  MacConkey  also  used  with  a 
similar  object  a  5  per  cent  glucose  bile-salt  bouillon  tinted  with  neutral 
litmus  as  in  Drigalski's  medium. 

With  reference  to  MacConkey's  fluid  media,  organisms  are  divided  into 
(1)  those  which  produce  both  acid  and  gas  ;  (2)  those  producing  acid  only  ; 
(3)  those  growing  but  not  producing  either  acid  or  gas  ;  (4)  those 
incapable  of  growing.  B.  coli  belongs  to  the  first  group  and  b.  typhosus 
to  the  second,  and  to  these  groups  also  belong  most  ordinary  organisms 
growing  in  faeces,  practically  none  of  which  are  found  in  the  third  and 
fourth  classes.  Thus  if  any  growth  takes  place  on  this  medium  when 
inoculated  with,  say,  water,  the  probability  is  that  the  bacteria  have  been 
derived  from  faeces,  but  of  course  their  identification  might  present  some 
difficulty.  With  the  neutral-red  solid  media  the  colonies  of  any  organism 
giving  rise  to  acid  will  be  of  a  beautiful  rose  red  colour. 

Petruschky's  Litmus  Whey. — The  preparation  of  this  medium,  which 
is  somewhat  difficult,  is  as  follows  : — Fresh  milk  is  slightly  warmed, 
and  sufficient  very  dilute  hydrochloric  acid  is  added  to  cause  precipita- 
tion of  the  casein,  which  is  now  filtered  off.  Dilute  sodium  hydrate 
solution  is  added  up  to,  but  not  beyond,  the  point  of  neutralisation,  and 
the  fluid  steamed  for  one  to  two  hours,  by  which  procedure  any  casein 
which  has  been  converted  into  acid  albumin  by  the  hydrochloric  acid 
is  precipitated.  This  is  filtered  off,  and  a  clear,  colourless,  perfectly 
neutral  fluid  should  result.  Its  chief  constituent,  of  course,  will  be 
lactose.  To  this  sufficient  Kubel-Tiemann  solution  of  litmus  is  added, 
the  medium  is  put  into  tubes  and  then  sterilised.  After  growth  has 
taken  place,  the  amount  of  acid  formed  can  be  estimated  by  dropping 
in  standardised  soda  solution  till  the  tint  of  an  uninoculated  tube  is 
reached. 

Media  for  growing  Trichophyta,  Moulds,  etc. — 1.  Beer  Wort  Agar. 
Take  beer  wort  as  obtainable  from  the  brewery  and  dilute  it  till  it  has  an 
s.g.  of  1100.  Add  1'5  per  cent  of  powdered  agar  and  heat  in  the  Koch 
till  it  is  dissolved  (usually  about  two  hours  are  necessary).  Filter 
rapidly  and  fill  into  tubes.  Sterilise  in  the  Koch  for  twenty  minutes  on 
three  successive  days.  If  the  medium  is  heated  too  long  it  loses  the 
capacity  of  solidifying. 

2.  Sabouraud's  medium  (modified).  Take  40  grammes  maltose  and 
10  grammes  Witte's  peptone  and  dissolve  these  in  one  litre  of  water,  then 
add  13  grammes  of  powdered  agar.  Heat  in  the  Koch  till  the  agar  is 
dissolved,  filter  and  fill  into  tubes,  sterilise  in  the  autoclave  for  twenty 
minutes  at  120°  C. 

To  use  these  for  isolating,  say,  the  Tinea  tonsurans,  pick  out  an 
infected  hair,  wash  in  absolute  alcohol  for  a  few  seconds,  then  wash  in 
changes  of  sterile  water  and  stab  the  hair  into  the  surface  of  the  medium 
in  a  number  of  places  ;  incubate  at  24°  C.  Usually  it  is  sufficient  to 
stab  the  hair  as  it  is  picked  from  the  skin  into  the  medium. 

Potatoes  as  Culture  Material. 

(a)  In  Potato  Jars. — The  jar  consists  of  a  round,  shallow, 
glass  vessel  with  a  similar  cover  (vide  Fig.  9).  It  is  washed 


POTATOES  AS  CULTURE  MATERIAL 


45 


FIG.  9.— Potato  jar. 


with  1-1000  corrosive  sublimate,  and  a  piece  of  circular  filter 

paper,  moistened  with  the  same,  is  laid  in  its  bottom.     On  this 

latter  are  placed  four  sterile  watch 

glasses.     Two  firm,  healthy,  small, 

round  potatoes,  as  free  from  eyes 

as  possible,  and  with  the  skin  whole, 

are   scrubbed    well   with   a,  brush 

under  the  tap  and  steeped  for  two 

or  three  hours  in  1-1000  corrosive 

sublimate.     They  are   steamed    in 

the    Koch's    steriliser     for    thirty 

minutes  or  longer,  or  in  the  auto- 
clave for  a  quarter  of  an  hour.  When 

cold,  each  is  grasped  between  the  left  thumb  and  forefinger 
(which  have  been  sterilised  with  sub- 
limate) and  cut  through  the  middle  with 
a  sterile  knife.  It  is  best  to  have  the 
cover  of  the  jar  raised  by  an  assistant, 
and  to  perform  the  cutting  beneath  it. 

FIG.  10.— Cylinder  of  potato    Each  nalf  is   Put   in   one   of    the    watch 
cut  obliquely.  glasses,    the    cut    surfaces, 

which   are   then    ready    for  ^,=,_ 

inoculation  with  a  bacterial  growth,  being  upper- 
most. Smaller  jars,  each  of  which  holds  half  of  a 
potato,  are  also  used  in  the  same  way  and  are  very 
convenient. 

(6)  By  Slices  in  Tubes. — This  method,  intro- 
duced by  Ehrlich,  is  the  best  means  of  utilising 
potatoes  as  a  medium.  A  large,  long  potato  is  well 
washed  and  scrubbed,  and  peeled  with  a  clean  knife. 
A  cylinder  is  then  bored  from  its  interior  with  an 
apple  corer  or  a  large  cork  borer,  and  is  cut  obliquely, 
as  in  Fig.  10.  Two  wedges  are  thus  obtained,  each 
of  which  is  placed  broad  end  downward  in  a  test-tube 
of  special  form  (see  Fig.  11).  In  the  wide  part  at 
the  bottom  of  this  tube  is  placed  a  piece  of  cotton 
wool,  which  catches  any  condensation  water  which 
may  form.  The  wedge  rests  on  the  constriction 
above  this  bulbous  portion.  The  tubes,  washed,  — Ehrlich's 

-,   .    i          i      •.,  ,  .      .,      ,     .  i  •      ji        tube  contain- 

dried,  and  with  cotton  wool  in  the  bottom  and  in  the   •        iece   of 
mouth,  are  sterilised  before  the  slices  of  potato  are   potato. 
introduced.     After  the  latter  are  inserted,  the  tubes 
are  sterilised  in  the  Koch  steam  steriliser  for  one  hour,  or  in 
the  autoclave  for  fifteen  minutes,  at  115°  C.     An  ordinary  test- 


FIG.  11. 


46     METHODS   OF   CULTIVATION   OF   BACTEKIA 

tube  may  be  used  with  a  piece  of  sterile  absorbent  wool  in  its 
bottom,  on  which  the  potato  may  rest. 

Glycerin  potato,  suitable  for  the  growth  of  the  tubercle 
bacillus,  may  be  prepared  by  covering  the  slices  in  the  tubes 
with  6  per  cent  solution  of  glycerin  in  water  and  steaming  for 
half  an  hour.  The  fluid  is  then  poured  off  and  the  sterilisation 
continued  for  another  half  hour. 

Potatoes  ought  not  to  be  prepared  long  before  being  used,  as 
the  surface  is  apt  to  become  dry  and  discoloured.  It  is  well  to 
take  the  reaction  of  the  potato  with  litmus  before  sterilisation, 
as  this  varies ;  normally  in  young  potatoes  it  is  weakly  acid. 
The  reaction  of  the  potato  may  be  more  accurately  estimated  by 
steaming  the  potato  slices  for  a  quarter  of  an  hour  in  a  known 
quantity  of  distilled  water  and  then  estimating  the  reaction  of 
the  water  by  phenol -phthaleine.  The  required  degree  of  acidity 
or  alkalinity  is  obtained  by  adding  the  necessary  quantity  of 
HC1  or  NaOH  solution  (p.  34)  and  steaming  for  other  fifteen 
minutes.  The  water  is  then  poured  off  and  sterilisation 
continued  for  another  half  hour.  Potatoes  before  being 
inoculated  ought  always  to  be  incubated  at  37°  C.  for  a  night, 
to  make  sure  that  their  sterilisation  has  been  successful. 

Eisner's  Medium. — This  is  one  of  the  media  introduced  in  the  study 
of  the  comparative  reactions  of  the  typhoid  bacillus  and  the  B.  coli. 
The  preparation  is  as  follows  :  500  grammes  potato  are  grated  up  in  a 
litre  of  water,  allowed  to  stand  over  night,  then  strained,  and  added  to 
an  equal  quantity  of  ordinary  15  per  cent  peptone  gelatin  which  has  not 
been  neutralised.  Normal  sodium  hydrate  solution  is  added  till  the 
reaction  is  feebly  acid  to  litmus,  the  whole  boiled  together,  filtered,  and 
sterilised.  Just  before  use  potassium  iodide  is  added  so  as  to  constitute 
one  per  cent  of  the  medium.  Moore  has  used  a  similar  agar  preparation. 
Here  500  grammes  potato  are  scraped  up  in  one  litre  of  water,  allowed  to 
stand  for  three  hours,  strained,  and  put  aside  over  night.  The  clear 
fluid  is  poured  off,  made  up  to  one  litre,  rendered  slightly  alkaline,  20 
grammes  agar  are  added,  and  the  whole  is  treated  as  in  making  ordinary 
agar.  The  medium  is  distributed  in  test-tubes — 10  c.c.  to  each — and 
immediately  before  use,  to  each  is  added  *5  c.c.  of  a  solution  of  10  grammes 
potassium  iodide  to  50  c.c.  water. 

Milk  as  a  Culture  Medium. 

This  is  a  convenient  medium  for  observing  the  effects  of 
bacterial  growth  in  changing  the  reaction,  in  coagulating  the 
soluble  albumin,  and  in  fermenting  the  lactose.  It  is  prepared 
as  follows  :  fresh  milk  is  taken,  preferably  after  having  had  the 
cream  "  separated  "  by  centrifugalisation,  as  is  practised  in  the 
best  dairies,  and  is  steamed  for  fifteen  minutes  in  the  Koch,  it 
is  then  set  aside  in  an  ice  chest  or  cool  place  over  night  to 


THE   USE   OF   THE   CULTURE   MEDIA          47 

facilitate  further  separation  of  cream.  The  milk  is  siphoned  off 
from  beneath  the  cream.  The  reaction  of  fresh  milk  is  alkaline. 
If  great  accuracy  is  necessary  any  required  degree  of  reaction 
may  be  obtained  by  the  titration  method.  It  is  then  placed  in 
tubes  and  sterilised  by  methods  B  (2)  or  B  (3). 

Bread  Paste. 

This  is  useful  for  growing  torulse,  moulds,  etc.  Some 
ordinary  bread  is  cut  into  slices,  and  then  dried  in  an  oven  till 
it  is  so  dry  that  it  can  be  pounded  to  a  fine  powder  in  a  mortar, 
or  rubbed  down  with  the  fingers  and  passed  through  a  sieve. 
Some  100  c.c.  flasks  are  washed,  dried,  and  sterilised,  and  a 
layer  of  the  powder  half  an  inch  thick  placed  on  the  bottom. 
Distilled  water,  sufficient  to  cover  the  whole  of  it,  is  then  run  in 
with  a  pipette  held  close  to  the  surface  of  the  bread,  and,  the 
cotton-wool  plugs  being  replaced,  the  flasks  are  sterilised  in  the 
Koch's  steriliser  by  method  B  (2).  The  reaction  is  slightly  acid. 

THE  USE  OF  THE  CULTURE  MEDIA. 

The  culture  of  bacteria  is  usually  carried  on  in  test-tubes 
conveniently  6  x  f  in.  These  ought  to  be  very  thoroughly 
washed  and  dripped,  and  their  mouths  plugged  with  plain 
cotton  wool.  They  are  then  sterilised  for  one  hour  at  170°  C. 
If  the  tubes  be  new,  the  glass,  being  usually  packed  in  straw, 
may  be  contaminated  with  the  extremely  resisting  spores  of 
the  b.  subtilis.  Cotton -wool  plugs  are  universally  used  for 
protecting  the  sterile  contents  of  flasks  and  tubes  from  con- 
tamination with  the  bacteria  of  the  air.  A  medium  thus 
protected  will  remain  sterile  for  years.  Whenever  a  protecting 
plug  is  removed  for  even  a  short  time,  the  sterility  of  the 
contents  may  be  endangered.  It  is  well  to  place  the  bouillon, 
gelatin,  and  agar  media  in  the  test- tubes  directly  after  filtration. 
The  media  can  then  be  sterilised  in  the  test-tubes. 

In  filling  tubes,  care  must  be  taken  to  run  the  liquid  down 
the  centre,  so  that  none  of  it  drops  on  the  inside  of  the  upper 
part  of  the  tube  with  which  the  cotton-wool  plug  will  be  in 
contact,  otherwise  the  latter  will  subsequently  stick  to 
the  glass  and  its  removal  will  be  difficult.  In  the  case  of 
liquid  media,  test-tubes  are  filled  about  one-third  full.  With 
the  solid  media  the  amount  varies.  In  the  case  of  gelatin 
media,  tubes  filled  one -third  full  and  allowed  to  solidify 
while  standing  upright,  are  those  commonly  used.  With 


48     METHODS    OF    CULTIVATION   OF   BACTERIA 


organisms  needing  an  abundant  supply  of  oxygen  the  best 
growth,  takes  place  on  the  surface  of  the  medium,  and  for 
practical  purposes  the  surface  ought  thus  to  be  as  large  as 
possible.  To  this  end  "  sloped "  agar  and  gelatin  tubes  are 
used.  To  prepare  these,  tubes  are  filled  only  about  one-sixth 
full,  and  after  sterilisation  are  allowed  to  solidify,  lying  on  their 
sides  with  their  necks  supported  so  that  the  contents  extend  3 


Fro.   13.— Tubes  of  media. 

a.    Ordinary  upright  tube.     b.   Sloped  tube, 
c.  "  Deep  "  tube  for  cultures  of  anaerobes. 


FIG.  12. — Apparatus  which 
may  be  used  for  filling  tubes. 
The  apparatus  explains  itself. 
The  india-rubber  stopper  with 
its  tubes  ought  to  be  sterilised 
before  use. 

to  4  inches  up,  giving  an  oblique  surface  after  solidification. 
Thus  agar  is  commonly  used  in  such  tubes  (less  frequently 
gelatin  is  also  "  sloped  "),  and  this  is  the  position  in  which  blood 
serum  is  inspissated.  Tubes,  especially  those  of  the  less  commonly 
used  media,  should  be  placed  in  large  jars  provided  with  stoppers, 
otherwise  the  contents  are  apt  to  evaporate.  A  tube  of  medium 
which  has  been  inoculated  with  a  bacterium,  and  on  which 
growth  has  taken  place,  is  called  a  "  culture."  A  "  pure  culture  " 
is  one  in  which  only  one  organism  is  present.  The  methods  of 


USE   OF   CULTURE   MEDIA  49 

obtaining  pure  cultures  will  presently  be  described.  When  a 
fresh  tube  of  medium  is  inoculated  from  an  already  existing 
culture,  the  resulting  growth  is  said  to  be  a  "  sub-culture  "  of  the 
first.  All  manipulations  involving  the  transference  of  small 
portions  of  growth  either  from  one  medium  to  another,  as  in  the 
inoculation  of  tubes,  or,  as  will  be  seen  later,  to  cover-glasses  for 
microscopic  examination,  are  effected  by  pieces  of  platinum  wire 
(Nos.  24  or  27  Birmingham  wire  gauge — the  former  being  the 
thicker)  fixed  in  glass  rods  8  inches  long.1  Every  worker  should 
have  three  such  wires.  Two  are  2£  inches  long,  one  of  these 
being  straight  (Fig.  14,  a),  and  the  other  having  a  loop  turned 
upon  it  (Fig.  14,  b).  .The  latter  is  referred  to  as  the  platinum 
"loop"  or  platinum  "eyelet,"  and  is  used  for  many  purposes. 


FIG.  14. — Platinum  wires  in  glass  handles. 

a.  Straight  needle  for  ordinary  puncture  inoculations.     &.  "  Platinum  loop." 
c.  Long  needle  for  inoculating  "  deep  "  tubes. 

"  Taking  a  loopf  ul "  is  a  phrase  constantly  used.  The  third  wire 
(Fig.  14,  c)  ought  to  be  4J  inches  long  and  straight.  It  is  used 
for  making  anaerobic  cultures.  It  is  also  very  useful  to  have 
at  hand  a  platinum-iridium  spud.  This  consists  of  a  piece  of 
platinum-iridium  about  1^  inches  long,  2  mm.  broad,  and  of 
sufficient  thickness  to  give  it  a  firm  consistence ;  its  distal  end  is 
expanded  into  a  diamond  shape,  and  its  proximal  is  screwed 
into  an  aluminium  rod.  It  is  very  useful  for  making  scrapings 
from  organs  and  for  disintegrating  felted  bacterial  cultures  ;  in 
such  manipulations  the  ordinary  platinum  wire  is  awkward  to 
work  with  as  it  bends  so  easily.  Cultures  on  a  solid  medium 
are  referred  to  (1)  as  "puncture"  or  "stab"  cultures  (German, 
Stichkultur),  or  (2)  as  "stroke"  or  "slant"  cultures  (Strichkultur), 
according  as  they  are  made  (1)  on  tubes  solidified  in  the  upright 
position,  or  (2)  on  sloped  tubes. 

1  Aluminium  rods  are  made  which  are  very  convenient.  The  end  is  split 
with  a  knife,  the  platinum  wire  is  inserted  and  fixed  by  pinching  the 
aluminium  on  it  in  a  vice. 


50     METHODS   OF   CULTIVATION   OF   BACTERIA 


To  inoculate,  say,  one  ordinary  upright  gelatin  tube  from 
another,  the  two  tubes  are  held  in  an  inverted  position  between 
the  forefinger  and  thumb  of  the  left  hand  with  their  mouths 
towards  the  person  holding  them ;  the  plugs  are  twisted  round 

once  or  twice,  to  make 
sure  they  are  not  adher- 
ing to  the  glass.  The 
short,  straight  platinum 
wire  is  then  heated  to 
redness  from  point  to 
insertion,  and  2  to  3 
inches  of  the  glass  rod 
are  also  passed  two  or 
three  times  through  the 
Bunsen  flame.  It  is  held 
between  the  right  fore 
and  middle  fingers,  with 
the  needle  projecting 
Fm.  15.— Another  method  of  inoculating  backwards,  i.e.  away 
solid  tubes.  from  the  right  palm. 

Remove  plug  from  cul- 
ture tube  with  right  forefinger  and  thumb,  and  continue  to  hold 
it  between  the  same  fingers,  by  the  part  which  projected  beyond 
the  mouth  of  the  tube.  Now  touch  the  culture  with  the  platinum 
needle,  and,  withdrawing  it,  replace  plug.  In  the  same  way 
remove  plug  from  tube  to  be  inoculated,  and  plunge  platinum 
wire  down  the  centre  of  the 
gelatin  to  within  half  an  inch 
of  the  bottom.  It  must  on 
no  account  touch  the  glass 
above  the  medium.  The  wire 
is  then  immediately  sterilised. 
A  variation  in  detail  of  this 
method  is  to  hold  the  plug 
of  the  tube  next  the  thumb 
between  the  fore  and  middle 
fingers,  and  the  plug  of  the 
other  between  the  middle  and 
ring  fingers,  then  to  make  the  inoculation  (Fig.  15).  If  a  tube 
contain  a  liquid  medium,  it  must  be  held  in  a  sloping  position 
between  the  same  fingers,  as  above.  For  a  stroke  culture  the 
platinum  loop  is  used,  and  a  little  of  the  culture  is  smeared  in 
a  line  along  the  surface  of  the  medium  from  below  upwards.  In 
inoculating  tubes,  it  is  always  well,  on  removing  the  plugs,  to 


FIG.  16. — Rack  for  platinum  needles. 


SEPARATION   OF   BACTERIA  51 

make  sure  that  no  strands  of  cotton  fibre  are  adhering  to  the  inside 
of  the  necks.  As  these  might  be  touched  with  the  charged  needle 
and  the  plug  thus  be  contaminated,  they  must  be  removed  by  heat- 
ing the  inoculating  needle  red-hot  and  scorching  them  off  with  it. 
When  the  platinum  wires  are  not  in  use  they  may  be  laid  in  a 
rack  made  by  bending  up  the  ends  of  a  piece  of  tin,  as  in 
Fig.  16.  To  prevent  contamination  of  cultures  by  bacteria 
falling  on  the  plugs  while  these  are  exposed  to  the  air  during 
inoculation  manipulations,  some  bacteriologists  singe  the  plugs 
in  the  flame  before  replacing.  This  is,  however,  in  most  cases  a 
needless  precaution.  If  the  top  of  a  plug  be  dusty  it  is  best  to 
singe  it  before  extraction. 

THE  METHODS  OF  THE  SEPARATION  OF  AEROBIC  ORGANISMS. 
PLATE  CULTURES. 

The  general  principle  underlying  the  methods  of  separation 
is  the  distribution  of  the  bacteria  in  one  of  the  solid  media 
liquefied  by  heat  and  the  dilution  of  the  mixture  so  that  the 
growths  produced  by  the  individual  bacteria — called  colonies — 
shall  be  suitably  apart.  In  order  to  render  the  colonies  easily 
accessible,  the  medium  is  made  to  solidify  in  as  thin  a  layer  as 
possible,  by  being  poured  out  on  glass  plates — hence  the  term 
"plate  cultures." 

As  the  optimum  temperature  varies  with  different  bacteria, 
it  is  necessary  to  use  both  gelatin  and  agar  media.  Many 
pathogenic  organisms,  e.g.  pneumococcus,  b.  diphtherise,  etc., 
grow  too  slowly  on  gelatin  to  allow  its  ready  use.  On  the  other 
hand,  many  organisms,  e.g.  some  occurring  in  water,  do  not 
develop  on  agar  incubated  at  37°  C. 

Separation  by  Gelatin  Media. — As  the  naked-eye  and  micro- 
scopic   appearances    of   colonies    are    often    very   characteristic, 
plate  cultures,  besides  use  in  separ- 
ation, are  often  taken  advantage  of 
in   the    description    of    individual 
organisms.        The      plate  -  culture 
method  can  also  be  used  to  test 
whether  a  tube  culture  is  or  is  not 
pure.       The  suspected  culture    is 
plated    (three    plates    being    pre- 
pared,   as    will    be   described).      If          FIG.  17.—  Petri's  capsule, 
all  the  colonies  are  the  same,  then      (Cover  shown  partially  raised.) 
the  cultures  may  be  held  to  be  pure. 

Either  simple  plates  of  glass  4  inches  by  3  inches  are  used, 


52     METHODS   OF   CULTIVATION   OF   BACTERIA 

or,  what  are  more  convenient,  circular  glass  cells  with  similar 
overlapping  covers.  The  latter  are  known  as  Petri's  dishes  or 
capsules  (Fig.  17).  They  are  usually  3  inches  in  diameter  and 
half  an  inch  deep.  The  advantage  of  these  is  that  they  do  not 
require  to  be  kept  level  by  a  special  apparatus  while  the  medium 
is  solidifying,  and  can  be  readily  handled  afterwards  without 
admitting  impurities.  Whether  plates  or  capsules  are  used, 
they  are  washed,  dried  with  a  clean  cloth,  and  sterilised  for  one 
hour  in  dry  air  at  170°  C.,  the  plates  being  packed  in  sheet-iron 
boxes  made  for  the  purpose  see  (Fig.  18). 

1.  Glass  Capsules. — While  in  certain  circumstances,  as  when 
the  number  of  colonies  has  to  be  counted,  it  is  best  to  use  plates 
of  glass ;  in  the  usual  laboratory  routine  Petri's  capsules  are  to 
be  preferred  for  the  above  reasons. 

The  contents  of  three  gelatin  tubes,  marked  a,  6,  c,1  are 
liquefied  by  placing  in  a  beaker  of  water  at  any  temperature 
between  25°  C.  and  38°  C.  Inoculate  a  with  the  bacterial 
mixture.  The  amount  of  the  latter  to  be  taken  varies,  and  can 
only  be  regulated  by  experience.  If  the  microscope  shows 
enormous  numbers  of  different  kinds  of  bacteria  present,  just  as 
much  as  adheres  to  the  point  of  a  straight  platinum  needle  is 
sufficient.  If  the  number  of  bacilli  is  small,  one  to  three  loops 
of  the  mixture  may  be  transferred  to  the  medium.  Shake  a 
well,  but  not  so  as  to  cause  many  fine  air-bubbles  to  form. 
Transfer  two  loops  of  gelatin  from  a  to  b.  Shake  b  and  transfer 
five  loops  to  c.  The  plugs  of  the  tubes  are  in  each  case  replaced 
and  the  tubes  returned  to  the  beaker.  The  contents  of  the 
three  tubes  are  then  poured  out  into  three  capsules.  In  doing 
so  the  plug  of  each  tube  is  removed  and  the  mouth  of  the  tube 
passed  two  or  three  times  through  the  Bunsen  flame,  the  tube 
being  meantime  rotated  round  a  longitudinal  axis.  Any  organ- 
isms on  its  rim  are  thus  killed.  The  capsules  are  labelled  and 
set  aside  till  growth  takes  place. 

For  accurate  work  it  will  be  found  convenient  to  carry  out 
the  dilutions  in  definite  proportions.  The  following  is  the  pro- 
cedure which  we  have  found  very  serviceable.  In  a  number  of 
small  sterile  test-tubes  '95  c.c.  sterile  water  is  put.  To  the  first 
tube  we  add  '05  c.c.  of  the  bacterial  mixture.  The  contents  of 
the  tube  are  well  shaken  up,  and  the  pipette  is  sterilised  by 
being  washed  out  with  boiling  water.  It  is  allowed  to  cool,  and 
•05  c.c.  of  fluid  is  transferred  from  the  first  tube  to  the  second. 
By  a  similar  procedure  '05  c.c.  is  transferred  from  the  second  to 

1  For  marking  glass  vessels  it  is  convenient  to  use  the  red,  blue,  or  yellow 
oil  pencils  made  for  the  purpose  by  Faber. 


KOCH'S   METHOD   OF   PLATE   CULTUKES       53 

the  third  and  so  on.  There  is  thus  effected  a  twenty-fold 
dilution  in  each  successive  tube.  After  these  steps  have  been 
carried  out  a  definite  amount,  say,  *05  c.c.  is  transferred  from 
each  tube  to  a  tube  of  melted  gelatine, — the  gelatine  being  after- 
wards plated  and  the  colonies  counted  when  growth  occurs. 
The  number  of  tubes  required  will  vary  according  to  the 
number  of  bacteria  in  the  original  mixture,  but  usually  four  or 
five  will  be  sufficient.  It  is  quite  evident  that  this  method  not 
only  enables  us  to  separate  bacteria,  but  if  necessary  gives  us  a 
means  of  estimating  exactly  the  number  in  the  original  mixture. 
The  colonies  appear  as  minute  rounded  points,  whitish  or 
variously  coloured.  Their  characters  can  be  more  minutely 
studied  by  means  of  a  hand-lens  or  by  inverting  the  capsule  on 
the  stage  of  a  microscope  and  examining  with  a  low  power 
through  the  bottom.  From  their  characters,  colour,  shape, 
contour,  appearance  of  surface,  liquefaction  or  non-liquefaction 
of  the  gelatin,  etc.,  the  colonies  can  be  classified  into  groups. 
Further  aid  in  the  grouping  of  the  varieties  is  obtained  by 
making  film  preparations  and  examining  them  microscopically. 
Gelatin  or  agar  tubes  may  then  be  inoculated  from  a  colony  of 
each  variety,  and  the  growths  obtained  are  then  examined  both 
as  to  their  purity  and  as  to  their  special  characters,  with  a  view 
to  their  identification  (p.  115). 

2.  Glass  Plates  (Koch). — When  plates  of  glass  are  to  be  used,  an 
apparatus  on  which  they  may  be  kept  level  while  the  medium  is  solidi- 
fying is,  as  has  been  said,  necessary.  An  apparatus  devised  by  Koch  is 
used  (Figs.  18,  19).  This  consists  of  a  circular  plate  of  glass  (with  the 
upper  surface  ground,  the  lower  polished)  on  which  the  plate  used  for 
pouring  out  the  medium  is  placed.  The  latter  is  protected  from  the  air 
during  solidification  by  a  bell  jar.  The  circular  plate  and  bell  jar  rest 
on  the  flat  rim  of  a  circular  glass  trough,  which  is  filled  quite  full  with 
a  mixture  of  ice  and  water,  to  facilitate  the  lowering  of  the  temperature 
of  whatever  is  placed  beneath  the  bell  jar.  The  glass  trough  rests  on 
corks  on  the  bottom  of  a  large  circular  trough,  which  catches  any  water 
that  may  be  spilled.  This  trough  in  turn  rests  on  a  wooden  triangle 
with  a  foot  at  each  corner,  the  height  of  which  can  be  adjusted,  and 
which  thus  constitutes  the  levelling  apparatus.  A  spirit  level  is  placed 
where  the  plate  is  to  go,  and  the  level  of  the  ground  glass  plate  thus 
assured.  There  is  also  prepared  a  "damp  chamber,"  in  which  the 
plates  are  to  be  stored  after  being  made.  This  consists  of  a  circular 
glass  trough  with  a  similar  cover.  It  is  sterilised  by  being  washed  out- 
side and  inside  with  perchlaride  of  mercury  1-1000,  and  a  circle  of  filter- 
paper  moistened  with  the  same  is  laid  on  its  bottom.  Glass  benches  on 
which  the  plates  may  be  laid  are  similarly  purified. 

To  separate  organisms  by  this  method  three  tubes,  a,  &,  c,  are  inocu- 
lated as  in  using  Petri's  capsules  (p.  52).  The  hands  having  been 
washed  in  perchloride  of  mercury  1-1000  and  dried,  the  plate  box  is 
opened,  and  a  plate  lifted  by  its  opposite  edges  and  transferred  to  the 


54     METHODS   OF   CULTIVATION   OF   BACTEEIA 

levelled  ground  glass  (as  in  Figs.  18,  19).  The  bell  jar  of  the  leveller 
being  now  lifted  a  little,  the  gelatin  in  tube  a  is  poured  out  on  the 
surface  of  the  sterile  plate,  and  while  still  fluid,  is  spread  by  stroking 


FIG.  18. — Koch's  levelling  apparatus  for  iise  in  preparing  plates. 
Hands  shown  in  first  position  for  transferring  sterile  plate  from  iron 
box  to  beneath  bell  jar,  where  it  subsequently  has  the  medium  poured 
out  upon  it. 

with  the  rim  of  the  tube.     After  the  medium  solidifies,  the  plate  is 
transferred  to  the  moist  chamber  as  rapidly  as  possible,  so  as  to  avoid 


FIG.  19. — Koch's  levelling  apparatus.  Hands  shown  in  second 
position  just  as  the  plate  is  lowered  on  to  the  ground  glass  surface. 
By  executing  the  transference  of  the  plate  from  the  box  in  this  way, 
the  surface  which  was  undermost  in  the  latter  is  uppermost  in  the 
leveller,  and  thus  never  meets  a  current  of  air  wliich  might  con- 
taminate it. 

atmospheric  contamination.     In  doing  this,  it  is  advisable  to  have  an 
assistant  to  raise  the  glass  covers.     Tubes  b  and  c  are  similarly  treated, 


SEPARATION   BY  AGAR   MEDIA 


55 


and  the  resulting  plates  stacked  in  series  on  the  top  of  a.  The  chamber 
is  labelled  and  set  aside  for  a  few  days  till  the  colonies  appear  on  the 
gelatin  plates.  The  further  procedure  is  of  the  same  nature  as  with 
Petri's  capsules. 

3.  Esmarch's  Roll  Tubes. — Here  the  principle  is  that  of 
dilution  as  before.  In  each  of  three  test-tubes  1J  or  1J  inch  in 
diameter,  gelatin  to  the  depth  of  f  of  an  inch  is  placed.  These 
are  sterilised.  The  gelatin  is  melted  and  in- 
oculated in  series  with  the  bacterial  mixture  as 
in  making  plate  cultures,  but  instead  of  being 
poured  out  it  is  rolled  in  a  nearly  horizontal 
position  under  a  cold  tap  or  on  a  block  of  ice  till 
it  solidifies  as  a  uniformly  thin  layer  on  the  inside 
of  the  tube.  Practically  we  deal  with  a  cylindrical 
sheet  of  gelatin  instead  of  a  flat  one.  A  con- 
venient form  of  tube  for  this  method  is  one  with 
a  constriction  a  short  distance  below  the  plug  of 
cotton  wool  (Fig.  20).  The  great  disadvantage  of 
the  method  is,  that  if  organisms  liquefying  the 
gelatin  be  present,  the  liquefied  gelatin  contamin- 
ates the  rest  of  the  medium. 

Separation  by  Agar  Media. — 1.  Agar  Plates. 
— The  only  difference  between  the  technique  here 
and  that  with  gelatin  depends  on  the  difference 
in  the  melting-points  of  the  two  media.  Agar, 
we  have  said,  melts  at  98°  C.,  and  becomes  again 
solid  a  little  under  40°  C.  As  it  is  dangerous 
to  expose  organisms  to  a  temperature  much 
above  42°  C.,  it  is  necessary  in  preparing  tubes 
of  agar  to  be  used  in  plate  cultures  to  first 
melt  the  agar,  by  boiling  in  a  vessel  of  water  for  a  few  minutes, 
and  then  to  cool  them  to  about  42°  C.  before  inoculating.  The 
manipulation  must  be  rapidly  carried  out,  as  the  margin  of 
time,  before  solidification  occurs,  is  narrow ;  otherwise  the 
details  are  the  same  as  for  gelatin.  Esmarch's  tubes  are  not 
suitable  for  use  here,  as  the  agar  does  not  adhere  well  to  the 
sides.  If  to  the  agar  2  per  cent  of  a  strong  watery  solution  of 
pure  gum  arabic  is  added,  Esmarch's  tubes  may,  however,  be 
used. 

2.  Separation  by  Stroking  Mixture  on  Surface  of  Agar 
Media. — The  bacterial  mixture,  instead  of  being  mixed  in  the 
medium,  is  spread  out  on  its  surface.  The  method  may  be  used 
both  when  the  bacteria  to  be  separated  are  in  a  fluid,  and  when 
contained  in  a  fairly  solid  tissue  or  substance,  such  as  a  piece 


FIG.  20. 
Esmarch's  tube 
for  roll  culture. 


56     METHODS   OF   CULTIVATION   OF   BACTERIA 

of  diphtheritic  membrane.  In  the  case  of  a  tissue,  for  example, 
a  small  portion  entangled  in  the  loop  of  a  platinum  needle  is 
stroked  in  successive  parallel  longitudinal  strokes  on  sloped 
agar,  the  same  aspect  being  brought  in  contact  with  the  agar  in 
all  the  strokes.  Three  strokes  may  be  made  on  each  tube,  and 
three  tubes  are  usually  sufficient.  In  this  process  the  organisms 
on  the  surface  of  the  tissue  are  gradually  rubbed  off,  and  when 
growth  has  taken  place  it  will  be  found  that  in  the  later 
strokes  the  colonies  are  less  numerous  than  in  the  earlier,  and 
sufficiently  far  apart  to  enable  parts  of  them  to  be  picked  off 
without  the  needle  touching  any  but  one  colony.  When,  as  in 
the  case  of  diphtheritic  membrane,  putrefactive  organisms  may 
be  present  on  the  surface  of  the  tissue,  these  can  be  in  great 
part  removed  by  washing  it  well  in  cold  water  previously 
sterilised  (vide  Diphtheria).  In  the  case  of  liquids,  the  loop  is 
charged  and  similarly  stroked.  Tubes  thus  inoculated  must  be 
put  in  the  incubator  in  the  upright  position  and  must  be 
handled  carefully  so  that  the  condensation  water,  which  always 
is  present  in  incubated  agar  tubes,  may  not  run  over  the  surface. 
Agar,  poured  out  in  a  Petri's  capsule  and  allowed  to  stand  till 
firm,  may  be  used  instead  of  successive  tubes.  Here  a  sufficient 
number  of  strokes  can  be  made  in  one  capsule.  Sloped  blood- 
serum  tubes  may  be  used  instead  of  agar.  The  method  is  rapid 
and  easy,  and  gives  good  results. 

Separation  of  Pathogenic  Bacteria  by  Inoculation  of 
Animals. — It  is  found  difficult  and  often  impossible  to  separate 
by  ordinary  plate  methods  certain  pathogenic  organisms,  such 
as  b.  tuberculosis,  b.  mallei,  and  the  pneumococcus,  when  such 
occur  in  conjunction  with  other  bacteria.  These  grow  best  on 
special  media,  and  the  first  two  (especially  the  tubercle  bacillus) 
grow  so  slowly  that  the  other  organisms  present  outgrow  them, 
cover  the  whole  plates,  and  make  separation  impossible.  The 
method  adopted  in  such  cases  is  to  inoculate  an  animal  with 
the  mixture  of  bacilli,  wait  until  the  particular  disease  develops, 
kill  the  animal,  and  with  all  aseptic  precautions  (vide  p.  123) 
inoculate  tubes  of  suitable  media  from  characteristic  lesions 
situated  away  from  the  seat  of  inoculation,  e.g.  from  spleen  in 
the  case  of  b.  tuberculosis,  spleen  or  liver  in  the  case  of  b. 
mallei,  and  heart  blood  in  the  case  of  pneumococcus. 

Separation  by  killing  Non-spored  Forms  by  Heat. — This  is 
a  method  which  has  a  limited  application.  As  has  been  said, 
the  spores  of  a  bacterium  resist  heat  more  than  the  vegetative 
forms.  When  a  mixture  contains  spores  of  one  bacterium  and 
vegetative  forms  of  this  and  other  bacteria,  then  if  the  mixture 


SEPARATION   OF  ANAEROBES  57 

be  boiled  for  a  few  minutes  all  the  vegetative  forms  will 
be  killed,  while  the  spores  will  remain  alive  and  will  develop 
subsequently.  This  method  can  be  easily  tested  in  the  case  of 
cultivating  b.  subtilis  from  hay  infusion.  A  little  chopped-up 
hay  is  placed  in  a  flask  of  water,  which  is  boiled  for  about  ten 
minutes.  On  this  being  allowed  to  cool  and  stand,  in  a  day  or 
two  a  scum  forms  on  the  surface,  which  is  found  to  be  a  pure 
culture  of  the  bacillus  subtilis.  The  method  is  also  often  used 
to  aid  in  the  separation  of  b.  tetani,  vide  infra. 


THE  PRINCIPLES  OF  THE  CULTURE  OF  ANAEROBIC 
ORGANISMS. 

All  ordinary  media,  after  preparation,  may  contain  traces  of 
free  oxygen,  and  will  absorb  more  from  the  air  on  standing.  (1) 
For  the  growth  of  anaerobes  this  oxygen  may  be  expelled  by 
the  prolonged  passing  of  an  inert  gas,  such  as  hydrogen,  through 
the  medium  (liquefied  if  necessary).  Further,  the  medium  must 
be  kept  in  an  atmosphere  of  the  same  gas,  while  growth  is  going 
on.  (2)  Media  for  anaerobes  may  be  kept  in  contact  with  the 
air,  if  they  contain  a  reducing  agent  which  does  not  interfere 
with  bacterial  growth.  Such  an  agent  takes  up  any  oxygen 
which  may  already  be  in  the  medium,  and  prevents  further 
absorption.  The  reducing  body  used  is  generally  glucose,  though 
formate  of  sodium  may  be  similarly  employed.  The  preparation 
of  such  media  has  already  been  described  (pp.  36,  37).  In  this 
case  the  medium  ought  to  be  of  considerable  thickness. 

The  Supply  of  Hydrogen  for  Anaerobic  Cultures. — The  gas  is  generated 
in  a  large  Kipp's  apparatus  from  pure  sulphuric  acid  and  pure  zinc.  It 
is  passed  through  three  wash-bottles,  as  in  Fig.  21.  In  the  first  is 
placed  a  solution  of  lead  acetate  (1  in  10  of  water)  to  remove  any  traces 
of  sulphuretted  hydrogen.  In  the  second  is  placed  a  1  in  10  solution  of 
silver  nitrate  to  remove  any  arsenietted  hydrogen  which  may  be  present 
if  the  zinc  is  not  quite  pure.  In  the  third  is  a  10  per  cent  solution 
of  pyrogallic  acid  in  caustic  potash  solution  (1  :  10)  to  remove  any 
traces  of  oxygen.  The  tube  leading  from  the  last  bottle  to  the  vessel 
containing  the  medium  ought  to  be  sterilised  by  passing  through  a 
Btmsen  flame,  and  should  have  a  small  plug  of  cotton  wool  in  it  to  filter 
the  hydrogen  germ-free. 

Separation  of  Anaerobic  Organisms. — (a)  By  Roll-tubes. — 
A  1J  inch  test-tube  has  as  much  gelatin  put  into  it  as  would  be 
used  in  the  Esmarch  roll- tube  method.  It  is  corked  with  an 
india-rubber  stopper  having  two  tubes  passing  through  it,  as  in 
Fig.  22.  The  ends  of  the  tubes  are  partly  drawn  out  as  shown, 


58     METHODS   OF   CULTIVATION   OF   BACTEEIA 

and  covered  with  plugs  of  cotton  wool.  Three  such  test-tubes 
are  prepared,  and  they  are  sterilised  in  the  steam  steriliser  (p.  27). 
After  sterilisation  the  gelatin  is  melted  and  one  tube  inoculated 
with  the  mixture  containing  the  anaerobes ;  the  second  is  inocu- 
lated from  the  first,  and  the  third  from  the  second,  as  in  making 
ordinary  gelatin  plates.  After  inoculation  the  gelatin  is  kept 
liquid  by  the  lower  ends  of  the  tubes  being  placed  in  water  at 
about  30°  C.,  and  hydrogen  is  passed  in  through  tube  x  for 
twenty  minutes.  The  gas -supply  tubes  are  then  completely 
sealed  off  at  x  and  i}  and  each  test-tube  is  rolled  as  in  Esmarch's 
method  till  the  gelatin  solidifies  as  a  thin  layer  on  the  internal 


FIG.  21. — Apparatus  for  supplying  hydrogen  for  anaerobic  cultures. 

«.  Kipp's  apparatus  for  manufacture  of  hydrogen,  b.  Wash-bottle  containing 
1-10  solution  of  lead  acetate,  c.  Wash-bottle  containing  1-10  solution  of  silver 
nitrate,  d.  Wash-bottle  containing  1-10  solution  of  pyrogallic  acid.  (b,  c,  and  d 
are  intentionally  drawn  to  a  larger  scale  than  a  to  show  details.) 

surface.  A  little  hard  paraffin  may  be  run  between  the  rim  of  the 
test-tube  and  the  stopper,  and  round  the  perforations  for  the  gas- 
supply  tubes,  to  ensure  that  the  apparatus  is  air-tight.  The 
gelatin  is  thus  in  an  atmosphere  of  hydrogen  in  which  the 
colonies  may  develop.  The  latter  may  be  examined  and  isolated 
in  a  way  which  will  be  presently  described.  The  method  is 
admirably  suited  for  all  anaerobes  which  grow  at  the  ordinary 
temperature. 

(b)  Bulloch's  Apparatus  for  Anaerobic  Culture. — This  can 
be  recommended  for  plating  out  mixtures  containing  anaerobes, 
and  for  obtaining  growths  (especially  surface  growths)  of  the 
latter.  It  consists  (Fig.  23)  of  a  glass  plate  as  base  on  which  a 
bell  jar  can  be  firmly  luted  down  with  unguentum  resinae.  In 


CULTUKE   OF   ANAEROBES 


59 


the  upper  part  of  the  bell  jar  are  two  apertures  furnished  with 

ground  stoppers,  and  through  each  of  the  latter  passes  a  glass  tube 

on  which  is  a  stop-cock.     One  tube,  bent  slightly  just  after 

passing   through   the   stopper,   extends 

nearly  to  the  bottom  of  the  chamber; 

the  other  terminates  immediately  below 

the  stopper.     In   using   the  apparatus 

there  is  set  on  the  base-plate  a  shallow 

dish,  of  slightly  less  diameter  than  that 

of  the  bell  jar,  and  having  a  little  heap 

of   from  two  to  four  grammes  of  dry 

pyrogallic  acid  placed  in  it  towards  one 

side.     Culture  plates  made  in  the  usual 

way  can  be  stacked  on  a  frame  of  glass 

rods  resting  on  the  edges  of  the  dish, 

or  a  beaker  containing  culture  tubes  can 

be  placed  in  it.     The  bell  jar  is  then 

placed  in  position  so  that  the  longer  glass   _ 
f  ,       .  P.,       FIG.    22.— Esmarchs    roll- 

tube  is  situated  over  that  part  of  the       tabe  adapted  for  cu]ture 

bottom  of  the  shallow  dish  farthest  away       containing  anaerobes. 

from  the  pyrogallic  acid,  and  the  bottom 

and  stoppers  are  luted.     The  air  in  the  bell  jar  is  now  expelled 

by  passing  a  current  of  hydrogen  through  the  short  glass-tube, 

and  both  stoppers  are  closed.  A 
partial  vacuum  is  then  effected  in 
the  jar  by  connecting  up  the  short 
tube  with  an  air-pump,  opening 
the  tap,  and  giving  a  few  strokes 
of  the  latter.  A  solution  of  109 
grms.  solid  caustic  potash  dissolved 
in  145  c.c.  water  is  made,  and 
into  the  vessel  containing  it  a 
rubber  tube  connected  with  the 
long  glass  tube  is  made  to  dip, 
and  the  stopper  of  the  latter 
being  opened,  the  fluid  is  forced 
into  the  chamber  and  spreads  over 
the  bottom  of  the  shallow  dish ; 

_____     potassium     pyrogallate     is     thus 

FIG.  23.-Bulloch's  apparatus  for    formed>    which    absorbs    an?    free 

anaerobic  plate  cultures.  oxygen  still  present.     Before  the 

whole   of   the  fluid   is  forced  in, 

the  rubber  tube  is  placed  in  a  little  boiled  water,  and  this,  passing 
through   the  glass  tubes,  washes   out  the  potash  and  prevents 


60     METHODS   OF   CULTIVATION   OF   BACTEKIA 

erosion  of  the  glass.  The  whole  apparatus  may  be  placed  in  the 
incubator  till  growth  occurs. 

It  is  often  advisable  in  dealing  with  material  suspected  to 
contain  anaerobes  to  inoculate  an  ordinary  deep  glucose  agar 
tube  with  it,  and  incubating  for  24  or  48  hours,  to  then  apply  an 
anaerobic  separation  method  to  the  resultant  growth.  Sometimes 
the  high  powers  of  resistance  of  spores  to  heat  may  be  taken 
advantage  of  in  aiding  the  separation  (vide  Tetanus). 

Cultures  of  Anaerobes. — When  by  one  or  other  of  the  above 
methods  separate  colonies  have  been  obtained,  growth  may  be 
maintained  on  media  in  contact  with  ordinary  air.  The  media 
generally  used  are  those  which  contain  reducing  agents,  and  the 
test-tubes  containing  the  medium  must  be  filled  to  a  depth  of  4 
inches.  They  are  sterilised  as  usual  and  are  called  "  deep  " 
tubes.  The  long  straight  platinum  wire  is  used  for  inoculating, 
and  it  is  plunged  well  down  into  the  "  deep  "  tube.  A  little  air 
gets  into  the  upper  part  of  the  needle  track,  and  no  growth  takes 
place  there,  but  in  the  lower  part  of  the  needle  track  growth 
occurs.  From  such  "  deep  "  cultures  growths  may  be  maintained 
indefinitely  by  successive  sub-cultures  in  similar  tubes.  Even 
ordinary  gelatin  and  agar  can  be  used  in  the  same  way  if  the 
medium  is  heated  to  boiling-point  before  use  to  expel  any 
absorbed  oxygen. 

Carroll's  Method  for  Anaerobic  Cultures. — This  may  be  used 
with  culture  tubes  containing  any  of  the  media  suitable  for 
anaerobes,  with  Esmarch's  roll-tubes,  or  with  fermentation  tubes. 
There  is  required  a  dry  tube  of  the  same  diameter  as  the  culture 
tube,  a  short  U-shaped  glass  tube,  and  two  pieces  of  rubber  tub- 
ing all  of  like  diameter.  The  culture  tube  having  been  inoculated, 
the  plug  is  pushed  home  below  the  lip  of  the  tube.  The  ends 
of  the  U-tube  are  smeared  with  vaseline  and  a  rubber  tube 
slipped  over  each ;  the  end  of  the  culture  tube  being  similarly 
treated,  the  free  end  of  one  of  the  rubber  tubes  is  pushed  over  it 
till  the  glass  of  the  U-tube  is  in  contact  with  the  glass  of  the 
culture  tube.  In  the  dry  tube  one  or  two  grammes  of  pyrogallic 
acid  are  placed  and  the  powder  is  packed  down  with  a  layer  of 
filter  paper.  Ten  or  twenty  cubic  centimetres  of  a  ten  per  cent 
solution  of  sodium  hydrate  are  then  poured  in  and  the  tube  is 
quickly  connected  up  by  the  rubber  tubing  with  the  other  end 
of  the  U-tube.  In  this  apparatus  the  oxygen  is  absorbed  by  the 
sodium  pyrogallate  and  the  conditions  for  anaerobic  growth 
are  fulfilled. 

Cultures  of  Anaerobes  in  Liquid  Media. — It  is  necessary  to 
employ  such  in  order  to  obtain  the  toxic  products  of  the  growth 


CULTURE   OF   ANAEROBES  IN   LIQUID   MEDIA     61 

of  anaerobes.  Glucose,  broth  is  most  convenient.  It  is  placed 
either  (1)  in  a  conical  flask  with  a  lateral  opening  and  a  perfor- 
ated india-rubber  stopper,  through  which  a  bent  glass  tube  passes, 
as  in  Fig.  24,  a,  by  which  hydrogen  may  be  delivered,  or  (2)  in 
a  conical  flask  with  a  rubber  stopper  furnished  with  two  holes, 
as  in  Fig.  24,  6,  through  a  tube  in  one  of  which  hydrogen  is 
delivered,  while  through  the  tube  in  the  other  the  gas  escapes. 
The  inner  end  of  the  gas  delivery  tube  must  in  either  case  be 
below  the  surface  of  the  liquid ;  the  inner  end  of  the  lateral 
nozzle  in  the  one  case,  and  the  inner  end  of  the  escape  tube  in 
the  other,  must  of  course  be  above  the  surface  of  the  liquid. 
The  single  tube  in  the  one  case  and  the  two  tubes  in  the  other 


FIG.  24. 

a.  Flask  for  anaerobes  in  liquid  media.     Lateral  nozzle  and  stopper  fitted  for 
hydrogen  supply.    6.  A  stopper  arranged  for  a  flask  without  lateral  nozzle. 

ought  to  be  partially  drawn  out  in  a  flame  to  facilitate  subsequent 
complete  sealing.  The  ends  of  the  tubes  through  which  the  gas 
is  to  pass  are  previously  protected  by  pieces  of  cotton  wool  tied 
on  them.  It  is  well  previously  to  place  in  the  tube,  through 
which  the  hydrogen  is  to  be  delivered,  a  little  plug  of  cotton 
wool.  The  flask  being  thus  prepared,  it  is  sterilised  by  methods 
B  (2)  or  B  (3).  On  cooling  it  is  ready  for  inoculation.  In  the 
case  of  the  flask  with  the  lateral  nozzle,  the  cotton-wool  covering 
having  been  momentarily  removed,  a  wire  charged  with  the 
organism  is  passed  down  to  the  bouillon.  In  the  other  kind  of 
flask  the  stopper  must  be  removed  for  an  instant  to  admit  the 
wire.  The  flask  is  then  connected  with  the  hydrogen  apparatus 
by  means  of  a  short  piece  of  sterile  india-rubber  tubing,  and 
hydrogen  is  passed  through  for  half  an  hour.  In  the  case  of 


62     METHODS   OF   CULTIVATION   OF   BACTERIA 


flask  (1),   the  lateral  nozzle  is  plugged   with   molten  paraffin 
covered  with  alternate  layers  of  cotton  wool  and  paraffin,  the 
whole  being  tightly  bound  on  with  string.     The  entrance  tube 
is    now  completely  drawn   off  in    the  flame  before  being  dis- 
connected from  the  hydrogen 
apparatus.     In   the   case    of 
flask  (2),  first  the  exit  tube 
and  then  the  entrance  tube 
are   sealed  off  in  the   flame 
before    the    flask    is    discon- 
nected   from    the    hydrogen 
apparatus.     It  is  well  in  the 
case    of   both   flasks  to   run 
some  melted  paraffin  all  over 
the   rubber  stopper.     Some- 
times much  gas  is  evolved  by 
anaerobes,    and    in    dealing 
with  an  organism  where  this 
FIG.  25.-Flask  arranged  for  culture  of     will  occur  prOvision  must  be 
anaerobes  which  develop  gas.  i      <•        •  rp,  . 

b  is  a  trough  of^rcmy  into  which  exit       is  conveniently  done  by  lead- 
ing down  the  exit  tube,  and 

letting  the  end  just  dip  into  a  trough  of  mercury  (Fig.  25), 
or  into  mercury  in  a  little  bottle  tied  on  to  the  end  of  the 
exit  tube.  The  pressure  of  gas  within  causes  an  escape  at  the 
mercury  contact,  which  at  the  same  time  acts  as  an  efficient 
valve.  The  method  of  culture  in  fluid  media  is  used  to  obtain 
the  soluble  products  of  such  anaerobes  as  the  tetanus  bacillus. 


FIG.  26. — Tubes  for  anaerobic  cultures  on  the  surface  of  solid  media. 

When  it  is  desired  to  grow  anaerobes  on  the  surface  of  a 
solid  medium  such  as  agar,  tubes  of  the  form  shown  in  Fig.  26, 
a  and  b,  may  be  used.  A  s'troke  culture  having  been  made,  the 
air  is  replaced  by  hydrogen  as  just  described,  and  the  tubes  are 
fused  at  the  constrictions.  Such  a  method  is  of  great  value 


HANGING-DROP   PREPARATIONS  63 

when  it  is  required  to  get  the  bacteria  free  from  admixture  of 
medium,  as  in  the  case  of  staining  flagella. 


MISCELLANEOUS  METHODS. 

Hanging-drop  Cultures. — It  is  often  necessary  to  observe 
micro-organisms  alive,  either  to  watch  the  method  and  rate  of 
their  multiplication,  or  to  investigate  whether  or  not  they  are 
motile.  This  is  effected  by  making  hanging-drop  cultures.  The 


FIG.  27. 

A.  Hollow-ground  slide  for  hanging-drop  cultures  shown  in  plan  and  section. 
B.  Another  form  of  slide  for  similar  cultures. 

method  in  the  form  to  be  described  is  only  suitable  for  aerobes. 
For  this  special  slides  are  necessary.  Two  forms  are  in  use  and 
are  shown  in  Fig.  27.  In  A  there  is  ground  out  on  one  surface 
a  hollow  having  a  diameter  of  about  half  an  inch.  That  shown 
in  B  explains  itself.  The  slide  to  be  used  and  a  cover-glass  are 
sterilised  by  hot  air  in  a  Petri's  dish,  or  simply  by  being  heated 
in  a  Bunsen  and  laid  in  a  sterile  Petri  to  cool.  In  the  case  of 
A,  one  or  other  of  two  manipulation  methods  may  be  employed. 
(1)  If  the  organism  be  growing  in  a  liquid  culture,  a  loop  of  the 
liquid  is  placed  on  the  middle  of  the  under  surface  of  the  sterile 
cover-glass,  which  is  held  in  forceps,  the  points  of  which  have 
been  sterilised  in  a  Bunsen  flame.  If  the  organism  be  growing 
in  a  solid  medium,  a  loopful  of  sterile  bouillon  is  placed  on  the 
cover-glass  in  the  same  position,  and  a  very  small  quantity  of 
the  culture  (picked  up  with  a  platinum  needle)  is  rubbed  up  in 


64     METHODS   OF   CULTIVATION   OF   BACTERIA 

the  bouillon.  The  cover  is  then  carefully  lowered  over  the  cell 
on  the  slide,  the  drop  not  being  allowed  to  touch  the  wall  or  the 
edge  of  the  cell.  The  edge  of  the  cover-glass  is  covered  with 
vaseline,  and  the  preparation  is  then  complete  and  may  be 
placed  under  the  microscope.  If  necessary,  it  may  be  first  in- 
cubated and  then  examined  on  a  warm  stage.  (2)  The  sterile 
cover-glass  is  placed  on  a  sterile  plate  (an  ordinary  glass  plate 
used  for  plate  cultures  is  convenient).  The  drop  is  then  placed 
on  its  upper  surface,  the  details  being  the  same  as  in  the  last 
case.  The  edge  of  the  cell  in  the  slide  is  then  painted  with 
vaseline,  and  the  slide,  held  with  the  hollow  surface  downwards, 
is  lowered  on  to  the  cover-glass,  to  the  rim  of  which  it  of  course 
adheres.  The  slide  with  the  cover  attached  is  then  quickly 
turned  right  side  up,  and  the  preparation  is  complete. 

In  the  case  of  B  the  drop  of  fluid  is  placed  on  the  centre  of 
the  table  x.  The  drop  must  be  thick  enough  to  come  in  contact 
with  the  cover-glass  when  the  latter  is  lowered  on  the  slide,  and 
not  large  enough  to  run  over  into  the  surrounding  trench  y. 
The  cover-glass  is  then  lowered  on  to  the  drop,  and  vaseline  is 
painted  along  the  margin  of  the  cover-glass.  The  method  of 
microscopic  examination  is  described  on  page  85. 

Anaerobic  Hanging-drop  Cultures. — The  growth  and  examination  of 
bacteria  in  hanging-drops  under  anaerobic  conditions  involve  consider- 


FIG.  28. — Graham  Brown's  chamber  for  anaerobic  hanging-drops. 
(A  portion  of  one  edge  of  upper  plate  is  shown  cut  away.) 

able  difficulty,  but  may  be  carried  out  in  an  apparatus  devised  by 
Graham  Brown  (Fig.  28).  It  consists  of  two  brass  plates  (a  and  a'} 
which  can  be  approximated  by  screws,  and  which  have  rounded 
apertures  in  their  middles  |  in.  in  diameter.  These  support  two  rubber 
rings,  an  upper  thinner  one  (6)  and  a  lower  thick  one  (d),  the  inner 
diameters  being  the  same  as  that  of  the  apertures  in  the  plates.  Between 


THE   COUNTING   OF   COLONIES  65 

b  and  d  is  placed  a  stout  cover-glass  of  suitable  size  (c)  ;  d  is  separated 
from  the  plate  of  by  a  square  plate  of  glass  (c)  (a  portion  of  an  ordinary 
glass-slide  for  microscopical  purposes  does  well).  Two  small  metal 
tubes  (/)  are  inserted  through  the  rubber  d.  Method  of  use  : — Fix  up 
the  apparatus  as  shown  above,  the  screws  being  just  tight  enough  to 
keep  the  parts  in  position,  and  sterilise  in  the  steam  steriliser.  Screw 
up  more  firmly  so  as  to  make  the  rubber  bulge  slightly.  Fill  a 
hypodermic  syringe  with  some  sterile  glucose  bouillon,  push  the  needle 
through  the  rubber  d,  and,  tilting  the  point  of  the  needle  against  the 
glass  c,  slowly  inject  enough  to  form  a  drop  on  the  under  surface  of  c. 
Withdraw  the  syringe  and  inoculate  its  point  with  the  bacterium,  again 
introduce  and  inoculate  the  drop.  Pass  hydrogen  througli  one  of  the 
tubes  for  fifteen  minutes,  close  the  ends  of  the  tubes,  and  incubate  at  the 
required  temperature.  The  apparatus  can  be  put  on  the  stage  of  a 
microscope  and  examined  from  time  to  time. 

The  Counting  of  Colonies. — An  approximate  estimate  of  the 
number  of  bacteria  present  in  a  given  amount  of  a  fluid  (say, 
water)  can  be  arrived  at 
by  counting  the  number 
of  colonies  which  develop 
when  that  amount  is 
added  to  a  tube  of  suit- 
able medium,  and  the 
latter  plated  and  incu- 
bated. An  ordinary  plate 
should  be  used  in  such  a 
case,  and  the  medium 
poured  out  in  as  rect- 
angular a  shape  as  pos- 
sible. For  the  counting,  FIG.  29. — Apparatus  for  counting  colonies, 
an  apparatus  such  as  is 

shown  in  Fig.  29  is  employed.  This  consists  of  a  sheet  of  glass 
ruled  into  squares  as  indicated,  and  supported  by  its  corners  on 
wooden  blocks.  The  table  to  which  these  blocks  are  attached 
has  a  dark  surface.  The  plate-culture  containing  the  colonies 
is  laid  on  the  top  of  the  ruled  glass.  The  numbers  of  colonies 
in,  say,  twenty  of  the  smaller  squares,  are  then  counted,  and  an 
average  struck.  The  total  number  of  squares  covered  by  the 
medium  is  then  taken,  and  by  a  simple  calculation  the  total 
number  of  colonies  present  can  be  obtained.  Plate-cultures  in 
Petri's  dishes  are  sometimes  employed  for  purposes  of  count- 
ing. The  bottoms  of  such  dishes  are,  however,  never,  flat,  and 
the  thickness  of  the  medium  thus  varies  in  different  parts. 
If  these  dishes  are  to  be  used,  a  circle  of  the  same  size  as  the 
dish  can  be  drawn  with  Chinese  white  on  a  black  card,  the 
circumference  divided  into  equal  arcs,  and  radii  drawn.  The 


66     METHODS   OF   CULTIVATION   OF   BACTERIA 


dish  is  then  laid  on  the  card,  the  number  of  colonies  in  a 
few  of  the  sectors  counted,  and  an  average  struck  as  before. 
In  counting  colonies  it  is  always  best  to  aid  the  eye  with  a  small 
hand-lens. 

Method  of  counting  Living  Bacteria  in  a  Culture. — This 
is  accomplished    by  putting  into  practice   a   dilution    method 

such  as  that  described  on  p.  52. 
Measured  amounts  of  high  dilutions 
are  plated,  and  the  numbers  of 
colonies  which  subsequently  develop 
are  counted.  In  applying  such  a 
method  it  is  necessary  to  have  pipettes  capable 
of  measuring  small  quantities  of  fluid.  Those 
discharging  -05  and  '1  c.c.  will  be  found  con- 
venient, and  such  pipettes  can  have  sub- 
divisions which  enable  them  to  be  used  for 
measuring  still  smaller  fractions  of  a  cubic 
centimetre.  Pipettes  of  this  kind  can  be 
obtained  at  the  instrument  makers.  Wright 
has  described  a  method  by  which  a  pipette 
(Fig.  30)  for  measuring  small  quantities  of 
fluid  can  be  made  from  ordinary  quill  tubing. 
The  method  is  as  follows : — A  piece  of  quill 
tubing  about  15  cm.  long  is  drawn  out  to  a 
capillary  stem.  A  standard  5  c.mm.  pipette 
(such  as  that  of  the  Gower's  hsemocytometer), 
or  the  pipette  described  later  on  p.  108,  is 
filled  with  mercury  and  the  metal  transferred 
to  the  capillary  stem  and  run  down  to  near 
its  extremity ;  the  upper  and  lower  limits  of 
the  mercury  are  marked  with  an  oil  pencil ; 

Flo^3°'~WrighA'sthe   mercury   is    then  displaced  up  the  tube 
250  c.mm.  pipette  ,.„  .,  •        i      T  ,    i        i  •  i 

fitted  with  nipple.  ™"  lts  previously  distal  end  is  at  the  proximal 
of  the  two  marks,  and  a  third  mark  is  made 
at  the  new  position  of  the  upper  end  of  the  droplet ;  the  mani- 
pulation is  repeated  three  more  times,  and  finally  the  tip  of 
the  tube  beyond  the  lowest  mark  is  broken  off.  Thus  on  the 
capillary  part  of  the  pipette  we  have  five  divisions,  each  cap- 
able of  holding  5  c.mm.  of  fluid.  The  rest  of  the  pipette  is  now 
calibrated-  so  as  to  determine  that  part  capable  of  containing 
225  c.mm.  and  250  c.mm.  This  is  done  by  placing  a  rubber 
nipple  on  the  wide  end  of  the  pipette  and  sucking  up  some 
water  tinted  with,  say,  methylene-blue  till  the  25  c.mm.  mark 
is  reached ;  a  small  air-bubble  is  then  allowed  to  enter  the 


250   c.  mm, 

225 


20 


15 


10 


2-5 


METHOD    OF   COUNTING   BACTERIA  67 

pipette,  then  other  25  c.mm.  of  fluid,  then  another  bubble,  and 
so  on  till  nine  volumes  each  of  25  c.mm.  have  been  sucked  up. 
A  mark  is  then  made  on  the  tube  at  the  upper  level  of  this 
amount,  other  25  c.mm.  are  sucked  up,  and  another  mark  made. 
The  fluid  is  expelled,  the  tube  dried,  and  that  part  containing 
the  225  and  250  marks  is  drawn  out  into  an  almost  capillary 
diameter,  the  manipulation  by  which  the  marks  were  originally 
arrived  at  is  repeated,  and  thus  in  the  new  marks  made  a  more 
accurate  calibration  for  these  amounts  is  attained.  In  order  to 
form  a  safety  chamber  a  second  bulb  is  formed  by  drawing  out 
the  tube  a  little  higher  up  as  in  the  figure,  and  finally  the  upper 
inch  or  two  are  bent  at  right  angles  to  the  calibrated  limb.  In 
doing  this  a  loop  may  be  thrown  on  the  plastic  melted  capillary 
tube  exactly  in  the  way  in  which  a  similar  loop  may  be  thrown 
on  a  piece  of  cord.  With  such  a  pipette  any  required  dilution 
of  a  culture  can  be  made  on  the  principles  already  described. 

Wright's  Method  of  counting  the  Bacteria  in  Dead 
Cultures. — In  the  making  of  vaccines  for  use  in  Wright's  pro- 
cedures it  is  necessary  to  know  the  total  number  of  bacterial 
cells,  whether  dead  or  living,  present  in  a  culture,  for  the  dead 
as  well  as  the  living  contain  the  toxins  which  may  stimulate 
the  therapeutic  capacities  of  the  body.  The  method  consists 
in  making  a  mixture  -of  blood  (whose  content  in  red  blood 
corpuscles  is  known)  with  the  bacterial  culture  and  comparing 
the  number  of  bacteria  with  the  number  of  corpuscles.  The 
observer  first  estimates  the  red  cells  in  his  blood ;  a  capillary 
pipette  with  a  rubber  nipple  and  with  a  mark  near  its  capillary 
extremity  is  then  taken,  blood  is  sucked  up  to  the  mark,  then 
an  air-bubble,  then  (according  to  the  empirical  estimate  the 
observer  forms  of  the  strength  of  his  bacterial  emulsion)  either 
one  volume  of  culture  and  three  volumes  of  diluting  fluid 
(e.g.  '85  per  cent  sodium  chloride)  or  two  of  culture  and  two 
of  fluid,  and  so  on ;  the  five  volumes  are  thoroughly  mixed  by 
being  drawn  backwards  and  forwards  in  the  wide  part  of  the 
pipette,  a  drop  is  then  blown  out  on  to  a  slide,  and  a  blood 
film  is  spread  which  may  be  stained  by  Irishman's  method. 
The  bacteria  and  blood-corpuscles  are  now  separately  enumerated 
in  a  series  of  fields  in  different  parts  of  the  preparation.  If 
a  dilution  has  been  taken  in  which  a  large  number  of  bacteria 
are  present,  an  artificial  field  may  be  used,  made  by  drawing 
with  the  oil  pencil  a  small  square  on  a  circular  cover-glass  and 
dropping  the  latter  on  to  the  diaphragm  of  the  microscope 
eye-piece.  Suppose,  now,  that  the  observer's  blood  contained 
5,000,000  red  cells  per  c.mm.,  that  one  volume  of  bacterial 


68     METHODS   OF   CULTIVATION   OF   BACTERIA 

emulsion  and  three  of  diluent  had  been  present  in  the  mixture, 
and  that  in  the  fields  examined  there  were  500  red  cells  and 
600  bacteria.  It  is  evident  that  in  the  undiluted  culture  for 
500  red  cells  there  would  have  been  2400  bacteria.  Now 
500:  2400::  5, 000,000:  24,000, 000,  which  last  figure  is  the 
number  of  bacteria  per  c.mm.  of  the  emulsion. 

The  Bacteriological  Examination  of  the  Blood. — (a)  This 
may  be  done  by  taking  a  small  drop  from  the  skin  surface,  e.g.  the 
lobe  of  the  ear.  The  part  should  be  thoroughly  washed  with 
1-1000  corrosive  sublimate  and  dried  with  sterile  cotton  wool. 
It  is  then  washed  with  absolute  alcohol  to  remove  the  antiseptic, 
drying  being  allowed  to  take  place  by  evaporation.  A  prick  is 
then  made  with  a  sterile  surgical  needle ;  the  drop  of  blood  is 
caught  with  a  sterile  platinum  loop  and  smeared  on  the  surface 
of  agar  or  blood  serum.  Film  preparations  for  microscopic 
examination  may  be  made  at  the  same  time.  It  is  rare  to  obtain 
growths  from  the  blood  of  the  human  subject  by  this  method 
(vide  special  chapters),  and  if  colonies  appear  the  procedure 
should  be  repeated  to  exclude  the  possibility  of  accidental  con- 
tamination. 

(b)  A  larger  quantity  of  blood  may  be  obtained  by  puncture 
of  a  vein ;  this  is  the  only  satisfactory  method,  and  should  be 
the  one  followed  whenever  practicable.  The  skin  over  a  vein 
in  the  forearm  or  on  the  dorsum  of  the  foot  having  been  sterilised, 
the  vein  is  made  turgid  by  pressure,  and  the  needle  of  a  syringe 
of  10-15  c.c.  capacity,  carefully  sterilised,  is  then  plunged 
obliquely  through  the  skin  into  the  lumen  of  the  vessel.  Several 
cubic  centimetres  of  blood  can  thus  be  withdrawn  into  the 
syringe.  Some  of  the  blood  (e.g.  1  c.c.)  should  be  added  to  small 
flasks  containing  50  c.c.  of  bouillon ;  the  rest  may  be  used  for 
smearing  the  surface  of  agar  tubes  or  may  be  added  to  melted 
agar  at  42°  C.,  which  is  then  plated.  The  flasks,  etc.,  are  then 
incubated.  By  this  method  cultures  c^h  often  be  obtained  where 
the  former  method  fails,  especially  in  severe  conditions  such  as 
ulcerative  endocarditis,  streptococcus  infection,  etc.  Part  of  the 
blood  may  be  incubated  by  itself  for  twenty-four  hours  and  cultures 
then  made. 

In  examining  the  blood  of  the  spleen  a  portion  of  the  skin 
over  the  organ  is  sterilised  in  the  same  way,  a  few  drops  are 
withdrawn  from  the  organ  by  a  sterile  hypodermic  syringe  and 
cultures  made.  (For  microscopic  methods,  vide  p.  87.) 

Bacteriological  Examination  of  the  Cerebro-spinal  Fluid — 
Lumbar  Puncture. — This  diagnostic  procedure,  which  is  some- 
times called  for  in  cases  of  meningitis,  can  be  carried  out  with 


EXAMINATION   OF   CEREBRO-SPINAL   FLUID    69 

a  sterilised  "antitoxin  needle  "  as  follows.  The  patient  should 
lie  on  the  right  side,  with  knees  somewhat  drawn  up  and  left 
shoulder  tilted  somewhat  forward,  so  that  the  back  is  fully 
exposed.  The  skin  over  the  lumbar  region  is  then  carefully 
sterilised,  as  above  described,  and  the  hands  of  the  operator  should 
be  similarly  treated.  The  spines  of  the  lumbar  vertebrae  having 
been  counted,  the  left  thumb  or  forefinger  is  pressed  into  the 
space  between  the  3rd  and  4th  spines  in  the  middle  line ;  the 
needle  is  then  inserted  about  half  an  inch  to  the  right  of  the 
middle  line  at  this  level  and  pushed  through  the  tissues,  its 
course  being  directed  slightly  inwards  and  upwards,  till  it  enters 
the  subdural  space.  When  this  occurs  fluid  passes  along 
the  needle,  sometimes  actually  spurting  out,  and  should  be 
received  in  a  sterile  test-tube.  Several  cubic  centimetres  of 
fluid  can  thus  usually  be  obtained,  no  suction  being  required ; 
thereafter  it  can  be  examined  bacteriologically  by  the  usual 
methods.  The  depth  of  the  subdural  space  from  the  surface 
varies  from  a  little  over  an  inch  in  children  to  three  inches,  or 
even  more,  in  adults — the  length  of  the  needle  must  be  suited 
accordingly.  In  making  the  puncture  it  is  convenient  to  have 
either  a  sterile  syringe  attached,  or  to  have  the  thick  end  of 
the  needle  covered  with  a  pad  of  sterile  wool,  which  is  of  course 
removed  at  once  when  the  fluid  begins  to  flow.  It  is  advisable 
to  use  the  platinum  needles  which  are  specially  made  for  the 
purpose,  as  a  sudden  movement  of  the  patient  may  snap  an 
ordinary  steel  needle. 

The  Bacteriological  Examination  of  Urine. — In  such  an 
examination  care  must  be  taken  to  prevent  the  contamination 
of  the  urine  by  extraneous  organisms.  In  the  male  it  is  usually 
sufficient  to  wash  thoroughly  the  glans  penis  and  the  meatus 
with  1-1000  corrosive  sublimate — the  lips  of  the  meatus  being 
everted  for  more  thorough  cleansing.  The  urine  is  then  passed 
into  a  series  of  sterile  flasks,  the  first  of  which  is  rejected  in 
case  contamination  has  occurred.  In  the  female,  after  similar 
precautions  as  regards  external  cleansing,  the  catheter  must  be 
used.  The  latter  must  be  boiled  for  half  an  hour,  and  anointed 
with  olive  oil  sterilised  by  half  au  hour's  exposure  in  a  plugged 
flask  to  a -temperature  of  120°  C.  Here,  again,  it  is  well  to 
reject  the  urine  first  passed.  It  is  often  advisable  to  allow  the 
urine  to  stand  in  a  cool  place  for  some  hours,  to  then  withdraw 
the  lower  portion  with  a  sterile  pipette,  to  centrifugalise  this, 
and  to  use  the  urine  in  the  lower  parts  of  the  centrifuge  tubes 
for  microscopic  examination  or  culture. 

Filtration  of  Cultures. — For  many  purposes  it  is  necessary 


70     METHODS   OF   CULTIVATION   OF   BACTERIA 


to  filter  all  the  organisms  from  fluids  in  which  they  may  have 
been  growing.     This  is  done  especially  in  obtaining  the  soluble 

toxic  products  of  bac- 
teria. The  only  filter 
capable  of  keeping  back 
such  minute  bodies  as 
bacteria  is  that  formed 
from  a  tube  of  unglazed 
porcelain  as  introduced 
by  Chamberland.  The 
efficiency  of  such  a  filter 
depends  on  the  fineness 
of  the  grain  of  the  clay 
from  which  it  is  made  ; 
the  finest  is  the  Kitasato 
filter  and  the  Chamber- 
land  "  B  "  pattern  ;  the 
next  finest  is  the  Cham- 
berland "F"  pattern, 
which  is  quite  good 
enough  for  ordinary 
FIG.  31. — Geissler's  vacuum  pump  arranged  with  work.  There  are  several 
manometer  for  filtering  cultures.  (The  tap  filters,  differing  slightly 
and  pump  are  intentionally  drawn  to  a  larger  •  -,  f  -i  11 
scale  than  the  manometer  board  to  show  J  dll»  au  Pos»esf>ing 

details.)  the  common  principle. 

Sometimes  the  fluid  is 

forced  through  the  porcelain  tube.  In  one  form  the  filter 
consists  practically  of  an  ordinary  tap  screwed  into  the  top 
of  a  porcelain  tube.  Through  the  latter  the  fluid  is  forced  and 
passes  into  a  chamber  formed  by  a 
metal  cylinder  which  surrounds  the 
porcelain  tube.  The  fluid  escapes 
by  an  aperture  at  the  bottom.  Such 
a  filter  is  very  suitable  for  domestic 
use,  or  for  use  in  surgical  operating- 
theatres.  As  considerable  pressure 
is  necessary,  it  is  evident  it  must  be 
put  on  a  pipe  leading  directly  from 
the  main.  Sometimes,  when  fluids 
to  be  filtered  are  very  albuminous,  FlG  32. -Chamberland' s  candle 
they  are  forced  through  a  porcelain  and  flask  arranged  for  filtration, 
cylinder  by  compressed  carbonic 

acid  gas.       For  ordinary  bacteriological  work,  filters  of  various 
kinds    are    in    the    market    (such    as    those    of     Klein    and 


THE   FILTRATION   OF   CULTURES 


71 


others),  but  the  most  generally  convenient  is  that  in  which 
the  fluid  is  sucked  through  the  porcelain  by  exhausting  the 
air  in  the  receptacle  into  which  it  is  to  flow.  This  is  con- 
veniently done  by  means  of  a  Geissler's  water-exhaust  pump 
(Fig.  31,  #),  which  must  be  fixed  to  a  tap  leading  directly  from 
the  main.  The  connection  with  the  tap  must  be  effected  by 
means  of  a  piece  of  thick- walled  rubber-tubing  as  short  as 
possible,  wired  on  to  tap  and  pump,  and  firmly  lashed  externally 
with  many  turns  of  strong  tape.  Before  lashing  with  the  tape 
the  tube  may  be  strengthened  by  fixing  round  it  with  rubber 
solution  strips  of  the  rubbered  canvas  used  for  mending  punctures 


FIG.  33. — Chambeiiand's  bougie 
arranged  with  lamp  funnel  for 
filtering  a  small  quantity  of 
fluid. 


FIG.  34.— Bougie  in- 
serted through 
rubber  stopper 
for  same  purpose 
as  in  Fig.  33. 


in  the  outer  cas«  of  a  bicycle  tyre.  A  manometer  tube  (6)  and 
a  receptacle  (c)  (the  latter  to  catch  any  back  flow  of  water  from 
the  pump  if  the  filter  accidentally  breaks)  are  intercepted  between 
the  filter  and  the  pump.  These  are  usually  arranged  on  a 
board  a,  as  in  Fig.  31.  Between  the  tube /and  the  pump  </, 
and  between  the  tube  d  and  the  filter,  it  is  convenient  to  insert 
lengths  of  flexible  lead-tubing  connected  up  at  each  end  with 
short,  stout-walled  rubber-tubing. 

Filters  are  arranged  in  various  ways,  (a)  An  apparatus  is 
arranged  as  in  Fig.  32.  The  fluid  to  be  filtered  is  placed  in  the 
cylindrical  vessel  a.  Into  this  a  "candle"  or  "bougie"  of 
porcelain  dips.  From  the  upper  end  of  the  bougie  a  glass  tube 
with  thick  rubber  connections,  as  in  Fig.  32,  proceeds  to  flask  b 


72     METHODS    OF   CULTIVATION   OF   BACTERIA 


and  passes  through  one  of  the  two  perforations  with  which  the 
rubber  stopper  of  the  flask  is  furnished.  Through  the  other 
opening  a  similar  tube  proceeds  to  the  exhaust-pump.  When 
the  latter  is  put  into  action  the  fluid  is  sucked  through  the 
porcelain  and  passes  over  into  flask  b.  This  apparatus  is  very 
good,  but  not  suitable  for  small  quantities  of  fluid. 

(6)  A  very  good  apparatus  can  be  arranged  with  a  lamp 
funnel  and  the  porcelain  bougie.  These  may  be  fitted  up  in 
two  ways.  (1)  An  india-rubber  washer  is  placed  round  the 
bougie  c  at  its  glazed  end  (vide  Fig.  33).  On  this  the  narrow 
end  of  the  funnel  d,  which  must,  of  course,  be  of  an  appropriate 
size,  rests.  A  broad  band  of  sheet  rubber  is  then  wrapped 

round  the  lower  end  of  the 
i  funnel,  and  the  projecting 

...'•*  part  of  the   bougie.     It  is 

firmly  wired  to  the  funnel 
above  and  to  the  bougie 
below.  The  extreme  point 
of  the  latter  is  left  exposed, 
and  the  whole  apparatus, 
being  supported  on  a  stand, 


—^'%     J 


FIG.  35. — Muencke's  modification  of 
Chamberland's  filter. 


is  connected  by  a  glass  tube 
with  the  lateral  tube  of  the 
flask  b ;  the  tube  a  is  con- 
nected with  the  exhaust- 
pump.  The  fluid  to  be 
filtered  is  placed  between 
the  funnel  and  the  bougie 
in  the  space  e,  and  is  sucked 
through  into  the  flask  b. 
(2)  This  modification  is  shown  in  Fig.  34.  Into  the  narrow  part 
of  the  funnel  an  india-rubber  bung  is  fitted,  with  a  perforation 
in  it  sufficiently  large  to  receive  the  candle,  which  it  should  grasp 
tightly. 

(c)  Muencke's  modification  of  the  Chamberland  filter  is 
seen  in  Fig.  35.  It  consists  of  a  thick-walled  flask  a,  the  lower 
part  conical,  the  upper  cylindrical,  with  a  strong  flange  on  the 
lip.  There  are  two  lateral  tubes,  one  horizontal  to  connect  with 
exhaust-pipe,  and  one  sloping,  by  which  the  contents  may  be 
poured  out.  Passing  into  the  upper  cylindrical  part  of  the  flask 
is  a  hollow  porcelain  cylinder  b,  of  less  diameter  than  the 
cylindrical  part  of  flask  a.  It  is  closed  below,  open  above,  and 
rests  by  a  projecting  rim  on  the  flange  of  the  flask,  an  asbestos 
washer,  c,  being  interposed.  The  fluid  to  be  filtered  is  placed 


THE   FILTRATION   OF   CULTURES 


73 


in  the  porcelain  cylinder,  and  the  whole  top  covered,  as  shown 
at  /,  with  an  india-rubber  cap  with  a  central  perforation ;  the 
tube   d   is   connected    with   the   exhaust-pump 
and  the  tube  e  plugged  with  a  rubber  stopper. 
When  a  large  quantity  of  fluid  is  to  be  filtered, 
a  receptacle  such  as  that  shown  in 
Fig.  36  may  be  used.     The  tap  in 
its  bottom  enables  the  filtrate  to  be 
removed    without    the    apparatus 
being  unshipped,  but  it  is  difficult 
to  get  the  tap  to  fit  so  accurately  as  not  to 
allow  air  to  pass  into  the  vacuum  chamber. 
For   filtering  small    quantities   of   fluid  the 
apparatus   shown   in   Fig.   37  may  be   used. 
It  consists  of   a  small   Chamberland  bougie 
fitted  by  a  rubber  tube  to  a  funnel,  the  stem 
of  which  has  been  passed 
through  a  rubber  cork ; 
this  cork  fits  into  a  tri- 
angular flask  with  side 
arm  for  connection  with 
exhaust. 

Before    any  one    of 
the  above  apparatus  is 
used,  it  ought  to  be  con- 
FIG.  36.— Flask  fitted  nected  up  as  far  as  pos- 
with    porcelain   sible   and    sterilised   in 
bougie  for  filtering    the      Koch,g      steriliser> 
large  quantities  of    m,  T         r 

flujj^  Ihe    ends    01    any    im- 

portant unconnected 
parts  ought  to  have  pieces  of  cotton  wool 
tied  over  them.  After  use  the  bougie  is 
to  be  sterilised  in  the  autoclave,  and  after 
being  dried  is  to  be  passed  carefully  through 
a  Bunsen  flame,  to  burn  off  all  organic 
matter.  If  the  latter  is  allowed  to  accumu- 
late the  pores  become  filled  up. 

The  success  of  filtration  must  be  tested 
by   inoculating  tubes  of   media  from    the 
filtrate,  and  observing  if  growth  takes  place,    FlG- 
as  there  may  be  minute  perforations  in  the 
candies  sufficiently  large  to  allow  bacteria 
to  pass  through.     Filtered  fluids  keep  for  a  long  time  if  the 
openings  of  the  glass  vessels  in  which  they  are  placed  are  kept 


37.  —  Flask  for 
filtering  small  quanti- 
ties of  fiuid. 


74     METHODS   OF   CULTIVATION   OF   BACTERIA 

thoroughly  closed,  and  if  these  vessels  be  kept  in  a  cool  place  in 
the  dark.  A  layer  of  sterile  toluol  about  half  an  inch  thick 
ought  to  be  run  on  to  the  top  of  the  filtered  fluid  to  protect  the 
latter  from  the  atmospheric  oxygen. 

Instead  of  being  filtered  off,  the  bacteria  may  be  killed  by 
various  antiseptics,  chiefly  volatile  oils,  such  as  oil  of  mustard 
(Roux).  These  oils  are  stated  to  have  no  injurious  effect  on  the 
chemical  substances  in  the  fluid,  and  they  may  be  subsequently 
removed  by  evaporation.  It  is  not  practicable  to  kill  the  bacteria 
by  heat  when  their  soluble  products  are  to  be  studied,  as  many 
of  the  latter  are  destroyed  by  a  lower  temperature  than  is  required 
to  kill  the  bacteria  themselves. 

Bacteria  can  be  almost  entirely  removed  from  fluid  cultures 
by  spinning  the  latter  in  a  centrifuge  of  very  high  speed  (e.g. 
C.  J.  Martin's  turbine  centrifuge),  and  this  method  is  sometimes 
adopted  in  practice. 

The  Observation  of  Bacterial  Fermentation  of  Sugars,  etc. 
— The  capacity  of  certain  species  of  bacteria  to  originate  fermenta- 
tions in  sugars  constitutes  an  important  biological  factor.  It 
is  well  to  consider  this  factor  in  relation  to  the  chemical  con- 
stitution of  the  sugars.  These  bodies  are  now  known  to  be  (to 
use  the  definition  of  Holleman)  aldehyde  or  ketone  alcohols 
containing  one  or  more  hydroxyl  groups,  one  of  which  is  directly 
linked  to  a  carbon  atom  in  union  with  carbonyl.  The  group 
characteristic  of  a  sugar.is  thus  -  CHOH  -  CO  -  .  The  sugars  are 
divided  into  monosaccharides  or  monoses,  disaccharides  (dioses), 
and  polysaccharides  (polyoses).  The  members  of  the  last  two 
groups  may  be  looked  on  as  derived  from  the  combination  of  two 
or  more  molecules  of  a  rnonosaccharide  with  the  elimination  of 
water  (e.g.  2C6H12O6  =  C12H22OU  +  H2O). 

Monosacc/iarides. — These  are  classified  according  to  the 
number  of  C  atoms  they  contain.  The  pentoses  ordinarily  used 
are  arabinose  (obtained  from  gum  arabic),  rhamnose  and  xylose 
(from  wood).  Among  the  hexoses  are  glucose  (dextrose)  with 
dextro-rotatory  properties.  Glucose  is  an  aldehyde  alcohol 
(aldose).  In  fruit  there  is  also  a  ketone  alcohol  (ketose)  called 
fructose,  which  from  its  laevo-rotatory  properties  is  also  known 
as  laevulose.  Other  hexoses  are  mannose  (from  the  vegetable 
ivory  nut)  and  galactose  (a  hydrolytic  derivative  of  lactose). 

Disaccharides  (C^H^On). — The  ordinary  members  of  this 
group  are  maltose  (derived  from  starch),  lactose,  and  cane  sugar 
(sucrose,  saccharose). 

Polysaccharides. — Examples  are  starch,  raffmose,  inulin  (from 
dahlia  roots),  dextrin,  arabin,  glycogen,  cellulose. 


BACTERIAL   FERMENTATION   OF   SUGARS       75 

If  we  consider  sugars  generally  from  the  point  of  view  of 
the  capacity  of  yeast  to  originate  alcoholic  fermentation  in  them, 
we  may  say  that  the  simpler  the  constitution  of  the  sugar  the 
more  easily  is  it  fermented.  Thus  the  monosaccharides  are 
more  easily  acted  on  by  yeast  than  the  di-  or  polysaccharides. 
Usually  an  independent  process  resulting  in  the  splitting  of  the 
higher  into  the  lower  is  preliminary  to  the  alcoholic  fermentation. 
Thus  yeast  first  inverts  cane  sugar  into  glucose  and  fructose  and 
then  acts  on  these  products.  From  what  is  known  it  is  probable 
that  similar  facts  hold  with  regard  to  the  action  of  bacteria. 

Besides  sugars  other  alcohols  with  large  molecules  may  be 
broken  down  by  bacterial  action,  and  these  bodies  have  been 
used  for  differentiating  the  properties  of  allied  bacteria.  Among 
these  substances  may  be  mentioned  the  trihydric  alcohol  glycerol 
(glycerin),  the  tetrahydric  erythritol  and  the  hexahydric  dulcitol 
(dulcite),  mannitol  (mannite),  and  sorbitol  (sorbite). 

Similarly  certain  glucosides,  such  as  salicin,  coniferin,  etc., 
have  been  used  for  testing  the  fermentative  properties  of 
bacteria.  Other  substances  allied  to  sugars  (e.g.  inosite)  have 
also  been  used. 

The  end  products  of  bacterial  fermentations  may  be  various. 
They  differ  according  to  the  sugar  employed  and  according  to 
the  species  of  bacterium  under  observation,  and  frequently  a  species 
which  will  ferment  one  sugar  has  no  effect  on  another.  The  sub- 
stances finally  produced,  speaking  roughly,  may  be  alcohols,  acids, 
or  gaseous  bodies  (chiefly  carbon  dioxide,  hydrogen,  and  methane). 
For  the  estimation  of  the  first  groups  complicated  chemical 
procedure  may  be  necessary.  The  tests  usually  employed  for 
the  detection  of  ordinary  fermentative  processes  depend  on  two 
kinds  of  changes,  namely  (a)  the  evolution  of  gases  and  (&)  the 
formation  of  acids.  Generally  speaking,  we  may  say  that  such 
tests  are  reliable  and  the  methods  to  be  pursued  are  simple. 
Besides  such  gases  as  those  named  some  organisms  give  rise 
to  sulphuretted  hydrogen  by  breaking  up  the  proteid.  The 
formation  of  this  gas  can  be  detected  by  the  blackening  of  lead 
acetate  when  it  is  added  to  the  gas-containing  medium. 

In  testing  the  effect  of  a  bacterium  on  a  given  sugar  it  is 
essential  that  this  sugar  alone  be  present ;  the  basis  of  the 
medium  ought  therefore  to  be  either  peptone  solution  (v.  p.  38) 
or  a  dextrose-free  bouillon  (v.  infra}.  The  sugar  or  other 
substance  is  added  in  the  proportion  of  from  a  half  to  one 
per  cent,  and  care  is  taken  not  to  overheat  during  sterilisation. 

To  obtain  a  "dextrose-free  "  bouillon  it  is  usual  to  inoculate  ordinary 
bouillon  with  some  organism,  such  as  b.  coli,  which  is  known  to  ferment 


76     METHODS   OF   CULTIVATION   OF   BACTERIA 

dextrose,  and  allow  the  latter  to  act  for  forty-eight  hours.  The  bouillon 
is  then  filtered  and  re-sterilised.  A  sample  is  tested  for  another  period  of 
forty-eight  hours  with  b.  coli,  to  make  certain  that  all  the  dextrose  has 
been  removed.  If  no  fresh  gas  -  formation  is  observed,  then  to  the 
remainder  of  the  bouillon  the  sugar  to  be  investigated  may  be  added. 
It  is  preferable  that  the  addition  should  be  made  in  the  form  of  a  sterile 
solution.  If  the  sugar  in  solid  form  be  placed  in  the  bouillon  and  this 
then  sterilised,  there  is  danger  that  chemical  changes  may  take  place 
in  the  sugar,  in  consequence  of  its  being  heated  in  the  presence  of 
substances  (such  as  the  alkali)  which  may  act  deleteriously  upon  it ;  in 
any  case  sterilisation  should  not  be  at  a  temperature  above  100°  C. 

For  the  observation  of  gas-formation  either  of  the  following 
methods  may  be  employed  : — 

(1)  Durham's  Tubes  (Fig.  38,  6).— The  plug  of  a  tube  which 
contains  about  one-third  more  than  usual  of  a  liquid  medium  is 


It  a  c 

FIG.   38. — Tubes  for  demonstrating  gas-formation  by  bacteria. 

a,  tube  with  "shake"  culture, 
fe,  Durham's  fermentation  tube, 
c,  ordinary  form  of  fermentation  tube. 

removed,  and  a  small  test-tube  is  slippsd  into  the  latter  mouth 
downwards.  The  plug  is  replaced  and  the  tube  sterilised  thrice 
for  ten  minutes  at  100°  C.  The  air  remaining  in  the  smaller 
tubs  is  thereby  expelled.  The  tube  is  then  inoculated  with  the 
bacterium  to  be  tested.  Any  gas  developed  collects  in  the  upper- 
part  of  the  inner  tube. 

(2)  The  Fermentation  Tube  (Fig.  38,  c). — This  consists  of  a 
tube  of  the  form  shown,  and  the  figure  also  indicates  the  extent 


BACTERIAL   FERMENTATION   OF   SUGARS       77 

to  which  it  ought  to  be  filled.  It  is  inoculated  in  the  bend  with 
the  gas-forming  organism,  and  when  growth  occurs  the  gas 
collects  in  the  upper  part  of  the  closed  limit,  the  medium  being 
displaced  into  the  bulb. 

For  the  observation  of  the  effect  of  an  organism  on  glucose 
the  following  method  may  be  employed  : — 

Gelatin  Shake  Cultures  (Fig.  38,  a). — The  gelatin  in  the  tube 
is  melted  as  for  making  plates ;  while  liquid  it  is  inoculated 
with  the  growth  to  be  observed,  and  shaken  to  distribute  the 
organisms  throughout  the  jelly.  It  is  then  allowed  to  solidify, 
and  is  set  aside  at  a  suitable  temperature.  If  the  bacterium  used 
is  a  gas-forming  one,  then,  as  growth  occurs,  little  bubbles  appear 
round  the  colonies. 

In  this  method  the  gas -formation  results  from  fermenta- 
tion of  the  glucose  naturally  present  in  the  medium  from 
transformation  of  the  glycogen  of  muscle.  The  amount  of 
glucose  naturally  present,  however,  varies  much,  and  therefore 
glucose  should  be  added  to  the  medium  if  the  effects  on  this 
sugar  are  to  be  observed  with  certainty.  The  shake  culture 
method  may  be  utilised  for  observing  fermentation  in  other 
sugars  by  adding  to  peptone  solution  containing  the  sugar 
10-15  per  cent  of  gelatin. 

The  development  of  an  acid  reaction  is  demonstrated  by  the 
addition  of  an  indicator  to  the  medium,  litmus  being  generally 
used.  The  details  of  composition  of  such  media  have  already 
been  given.  In  Hiss's  serum  water  media  the  production  of 
acid  also  leads  to  coagulation  of  the  medium.  Sometimes  acid 
is  formed  very  slowly  from  sugars,  so  that  it  is  well  to  keep  the 
cultures  under  observation  for  several  days. 

Acid  and  gas-formation  may  be  simultaneously  tested  for,  by 
placing  the  fluid  medium  containing  the  indicator  in  Durham's 
tubes. 

In  all  tests  in  which  sugars  are  used  a  control  uninoculated 
tube  ought  to  be  incubated  with  the  bacterial  cultures,  as  changes 
in  reaction  sometimes  spontaneously  occur  in  media  containing 
unstable  sugars. 

The  capacity  of  an  organism  to  produce  acid  may  be  measured 
by  taking  a  standard  amount  of  a  fluid  medium  and  allowing 
growth  to  take  place  for  a  standard  time,  and  then  adding  an 
amount  of,  say,  decinormal  soda  solution  sufficient  to  bring  the 
litmus  back  to  the  tint  of  the  original  medium. 

The  Observation  of  Indol-formation  by  Bacteria. — The 
formation  of  indol  from  albumin  by  a  bacterium  sometimes  con- 
stitutes an  important  specific  characteristic.  To  observe  indol 


78     METHODS   OF   CULTIVATION    OF   BACTERIA 

production  the  bacterium  is  grown,  preferably  at  incubation 
temperature,  in  a  fluid,  medium  containing  peptone.  The  latter 
may  either  be  ordinary  bouillon  or  preferably  peptone  solution 
(see  p.  38).  Indol  production  is  recognised  by  the  fact  that  when 
acted  on  by  nitric  acid  in  the  presence  of  nitrites,  a  nitroso-indol 
compound  is  produced,  which  has  a  rosy  red  colour.  Some 
bacteria  (e.g.  the  cholera  vibrio)  produce  nitrites  as  well  as  indol, 
but  usually  in  making  the  test  (e.g.  in  the  case  of  b.  coli)  the 
nitrites  must  be  added.  This  is  effected  by  adding  to  an  ordinary 
tube  of  medium  1  c.c.  of  a  *02  per  cent  solution  of  potassium 
nitrite,  and  testing  with  pure  nitric  or  sulphuric  acid.  In  any 
case  only  a  drop  of  the  acid  need  be  added  to,  say,  10  c.c.  of 
medium.  If  no  result  be  obtained  at  once  it  is  well  to  allow 
the  tube  to  stand  for  an  hour,  as  sometimes  the  reaction  is  very 
slowly  produced.  In  many  instances  incubation  at  37°  C.  for 
several  days  may  be  necessary  before  the  presence  of  indol  is 
demonstrable.  The  amount  of  indol  produced  by  a  bacterium 
seems  to  vary  very  much  with  certain  unknown  qualities  of  the 
peptone.  It  is  well  therefore  to  test  a  series  of  peptones  with 
an  organism  (such  as  the  b.  coli)  known  to  produce  indol,  and 
noting  the  sample  with  which  the  best  reaction  is  obtained,  to 
reserve  it  for  making  media  to  be  used  for  the  detection  of  this 
product. 

The  Drying  of  Substances  in  vacuo. — As  many  substances, 
for  example  toxins  and  antitoxins,  with  which  bacteriology  is 
concerned  would  be  destroyed  by  drying  with  heat  as  is  done  in 
ordinary  chemical  work,  it  is  necessary  to  remove  the  water  at 
the  ordinary  room  temperature.  This  is  most  quickly  effected 
by  drying  in  vacuo  in  the  presence  of  some  substance  such  as 
strong  sulphuric  acid  which  readily  takes  up  water  vapour.  The 
vacuum  produced  by  a  water-pump  is  here  not  available,  as  in 
such  a  vacuum  there  must  always  be  water  vapour  present.  An 
air-pump  is  therefore  to  be  employed.  Here  we  have  found  the 
Geryk  pump  most  efficient,  and  it  has  this  further  advantage, 
that  its  internal  parts  are  lubricated  with  an  oil  of  very  low 
vapour  density  so  that  almost  a  perfect  vacuum  is  obtainable. 
The  apparatus  is  shown  in  Fig.  39.  The  vacuum  chamber 
consists  of  a  bell-jar  set  on  a  brass  plate.  A  perforation  in  the 
centre  of  the  latter  leads  into  the  pipe  a,  which  can  be  connected 
by  strong-walled  rubber-tubing  with  the  air-pump,  and  which 
can  be  cut  off  from  the  latter  by  a  stop-cock  b.  In  using  the 
apparatus  the  substance  to  be  dried  is  poured  out  in  flat  dishes 
(one-half  of  a  Petri  capsule  does  very  well),  and  these  are  stacked 
alternately  with  similar  dishes  of  strong  'sulphuric  acid  on  a 


STORING   AND   INCUBATION   OF   CULTURES     79 

stand  which  rests  on  the  brass  plate.  The  edge  of  the  bell-jar 
is  well  luted  with  unguentum  resinae  and  placed  in  position  and 
the  chamber  exhausted.  In  a  few  hours,  if,  as  is  always  advis- 
able, each  dish  have  contained  only  a  thin  layer  of  fluid,  the 
drying  will  be  complete.  The  vacuum  is  then  broken  by 
admitting  air  very  slowly  through  a  bye-pass  c,  and  the  bell-jar 
is  removed.  In  such  an  apparatus  it  is  always  advisable,  as  is 
shown  in  the  figure,  to  have  interposed  between  the  pump  and 
the  vacuum  chamber  a  Wolffs  bottle  containing  sulphuric  acid. 
This  protects  the  oil  of  the  pump  from  contamination  with 


FIG.  39. — Geryk  air-pump  for  drying  in  vacuo. 

water  vapour.  Whenever  the  vacuum  is  produced  the  rubber- 
tube  should  be  at  once  disconnected  from  a,  the  cock  b  being 
shut.  It  is  advisable  when  the  apparatus  is  exhausted  to  cover 
the  vacuum  chamber  and  the  Wolffs  bottle  with  wire  guards 
covered  with  strong  cloth,  in  case,  under  the  external  pressure, 
the  glass  vessels  give  way. 

The  Storing  and  Incubation  of  Cultures. — Gelatin  cultures 
must  be  grown  at  a  temperature  below  their  melting-point,  i.e. 
for  10  per  cent  gelatin,  below  22°  C.  They  are  usually  kept  in 
ordinary  rooms,  which  vary,  of  course,  in  temperature  at  different 
times,  but  which  have  usually  a  range  of  from  about  12°  C.  to 
18°  C.  Agar  and  serum  media  are  usually  employed  to  grow 
bacteria  at  a  higher  temperature,  corresponding  to  that  at  which 


80     METHODS    OF    CULTIVATION    OF   BACTERIA 

the  organisms  grow  best,  usually  37°  C.  in  the  case  of 
pathogenic  organisms.  For  the  purpose  of  maintaining  a  uniform 
temperature  incubators  are  used,  These  vary  much  in  the 
details  of  their  structure,  but  all  consist  of  a  chamber  with 
double  walls  between  which  some  fluid  (water  or  glycerin  and 
water)  is  placed,  which,  when  raised  to  a  certain  temperature, 
ensures  a  fairly  constant  distribution  of  the  heat  round  the 
chamber.  The  latter  is  also  furnished  with  double  doors,  the 
inner  being  usually  of  glass.  Heat  is  supplied  from  a  burner 
fixed  below.  These  burners  vary  much  in 
gr  design.  Sometimes  a  mechanism  devised  in 

...- •:---....  Koch's   laboratory   is   affixed,   which   auto- 

a  matically  turns  off  the  gas  if  the  light  be 
accidentally  extinguished.  Between  the  tap 
supplying  the  gas,  and  the  burner,  is  inter- 
posed a  gas  regulator.  Such  regulators 
vary  in  design,  but  for  ordinary  chambers 
which  require  to  be  kept  at  a  constant  tem- 
perature, Reichert's  is  as  good  and  simple 
as  any  and  is  not  expensive.  It  is  shown 
in  Fig.  40. 


It  consists  of  a  long  tube /closed  at  the  lower 
end,  open  at  the  upper,  and  furnished  ^ith  two 
lateral  tubes.  The  lower  part  is  filled  with 
mercury  up  to  a  point  above  the  level  of  the  lower 
lateral  tube.  The  end  of  the  latter  is  closed  by  a 
brass  cap  through  which  a  screw  d  passes,  the 
inner  end  of  which  lies  free  in  the  mercury.  The 
_  height  of  the  latter  in  the  perpendicular  tube  can 

J  thus  be  varied   by  increasing  or  decreasing  the 

capacity  of  the  lateral  tube  by  turning  the  screw 
a  few  turns  out  of  or  into  it.     Into  the  upper 
open  end  of  the  perpendicular  tube  fits  accurately 
FIG.  40. — Reichert's      a  bent  tube  g,  drawn  out  below  to  a  comparatively 
gas  regulator.  small   open    point  c,   and   having   in    its   side  a 

little    above    the    point    a    minute    needle-hole 

called  the  peephole  or  bye-pass  e.  To  fix  the  apparatus  the  long 
mercury  bulb  is  placed  in  the  jacket  of  the  chamber  to  be  controlled, 
tube  a  is  connected  to  gas  supply,  tube  b  with  the  burner.  The  upper 
level  of  the  mercury  should  be  some  distance  below  the  lower  open  end 
of  tube  c.  The  burner  is  now  lit.  The  gas  passes  in  at  a  through  c 
and  e  and  out  at  b  to  the  burner.  When  the  thermometer  in  the 
interior  of  the  chamber  indicates  that  the  desired  temperature  has  been 
reached,  the  screw  d  is  turned  till  the  mercury  reaches  the  end  of  the 
tube  c.  Gas  can  only  now  pass  through  the  peephole  e,  and  the  flame 
goes  down.  The  contents  of  the  jacket  cool,  the  mercury  contracts  off 
the  end  of  tube  c,  and  the  flame  rises.  This  alternation  going  on,  the 
temperature  of  the  chamber  is  kept  very  nearly  constant.  If  the  mercury 
cuts  off  the  gas  supply  before  the  desired  temperature  is  reached,  and 


STORING   AND   INCUBATION    OF   CULTURES     81 

the  screw  d  is  as  far  out  as  it  will  go,  then  some  of  the  mercury  must  be 
removed.  Similarly,  if  when  the  desired  temperature  is  reached  and  the 
screw  d  is  as  far  in  as  it  can  go,  the  mercury  does  not  reach  c,  some  more 
must  be  introduced.  If  the  amount  of  gas  which  passes  through  the 
peephole  is  sufficient  still  to  raise  the  temperature  of  the  chamber  when 
c  is  closed  by  the  rise  of  the  mercury,  then  the  peephole  is  too  large.  Tube 
c  must  be  unshipped  and  e  plastered  over  with  sealing-wax,  which  is 
pricked,  while  still  soft,  with  a  very  fine  needle.  The  gas  flame,  when 
only  the  peephole  is  supplying  gas,  ought  to  be  sufficiently  large  not  to 
be  blown  out  by  small  currents  of  air.  If  the  pressure  of  gas  supplied  to  a 
regulator  varies  much  in  the  24  hours  a  pressure  regulator  ought  to  be 
interposed  between  the  gas  tap  and  the  instrument.  Several  varieties  of 
these  can  be  obtained.  In  all  cases  g  ought  to  be  fixed  to  b  with  a  turn 
of  wire. 


FIG.  41. — Hearson's  incubator  for  use  at  37°  C. 

The  varieties  of  incubators  are,  as  we  have  said,  numerous. 
The  most  complicated  and  expensive  are  made  by  German 
manufacturers.  Many  of  these  are  unsatisfactory.  They  easily 
get  out  of  order  and  are  difficult  to  repair.  We  have  found 
those  of  Hearson  of  London  extremely  good,  and  in  proportion 
to  their  size  much  cheaper  than  the  German  articles.  They  are 
fitted  with  an  admirable  regulator.  It  is  preferable  in  using  an 
incubator  to  connect  the  regulator  with  the  gas  supply  and  with 
the  Bunsen  by  flexible  metal  tubing.  It  is  necessary  to  see  that 
there  is  not  too  much  evaporation  from  the  surface  of  cultures 
placed  within  incubators,  otherwise  they  may  quickly  dry  up. 
It  is  thus  advisable  to  raise  the  amount  of  water  vapour  in  the 
interior  by  having  in  the  bottom  of  the  incubator  a  flat  dish  full 
6 


82     METHODS    OF   CULTIVATION    OF   BACTERIA 

of  water  from  which  evaporation  may  take  place.  Tubes  which 
will  require  to  be  long  in  the  incubator  should  have  their  plugs 
covered  either  by  india-rubber  caps  or  by  pieces  of  sheet  rubber 
tied  over  them.  These  caps  should  be  previously  sterilised  in 
1-1000  corrosive  sublimate  and  then  dried.  Before  they  are 
placed  on  the  tubes  the  cotton-wool  plug  ought  to  be  well  singed 
in  a  flame.  "  Cool "  incubators  are  often  used  for  incubating 
gelatin  at  21°  to  22°  C.  An  incubator  of  this  kind  fitted  with  a 
low-temperature  Hearson's  regulator  is  in  the  market. 

Method  of  mounting  Bacterial  Cultures  as  Permanent 
Museum  Specimens.  (Richard  Muir).  —  (a)  Stab  or  stroke 
cultures  in  nutrient  gelatin  or  agar  media.  — When  the  culture 
shows  typical  characters,  further  growth  is  arrested  by  placing 
tube  in  a  formol  vapour  chamber,  or  by  saturating  the  cotton- 
wool plug  with  strong  formalin.  Then  leave  for  a  day  or  two. 
Make  up  following  : — 

(1)  Thymol  Water  (saturated  in  cold).         .         .  100  c.c. 

Glycerin 20  c.c. 

Acetate  of  Potasli  .         .         .         .         .  5  grams. 

Coignet's  (gold  label)  Gelatin          ...  10  grams. 

Render  the  mixture  acid  to  litmus  with  acetic  acid  ;  clear  with  white 
of  egg  and  filter. 

Warm  to  about  40°  C.,  and  removing  cotton-wool  plug  from 
culture  take  a  little  of  the  preserving  fluid  in  a  pipette  and 
allow  to  run  gently  over  surface  of  medium  in  tube.  Place  in 
such  a  position  that  a  thin  layer  of  the  preserving  medium 
remains  completely  covering  the  growth  and  the  surface  of 
culture  medium.  The  gelatin  is  now  allowed  to  solidify.  Add 
three  or  four  drops  of  strong  formalin  to  the  tube  and  fill  up  to 
within  a  quarter  of  an  inch  of  the  top  of  the  tube  with  the 
following  fluid  : — 

(2)  Thymol  Water  (saturated  in  cold) .         .         .         100  c.c. 
Glycerin          .         .         .         .         .         .         .         20  c.c. 

Acetate  of  Potash  ......         5  grams. 

Cover  top  of  tube  with  a  small  piece  of  paper  so  as  to  keep 
out  dust,  allow  to  stand  for  a  day  or  two  so  that  small  air-bells 
may  rise  to  the  surface. 

To  seal  tube,  pour  melted  paraffin  gently  on  to  the  surface 
of  fluid  to  near  the  top  of  tube  ;  allow  to  solidify.  Cover  paraffin 
with  layer  of  alcoholic  orange  shellac  cement ;  allow  this  to  set 
and  repeat  until  the  cement  becomes  level  with  top  of  test  tube. 
When  set,  a  few  drops  of  black  lacquer  are  put  on  and  a  circular 
cover-glass  of  about  the  same  diameter  as  the  mouth  of  tube  is 
placed  so  as  completely  to  seal  it. 


GENERAL  LABORATORY  RULES       83 

(b)  The  following  method  is  useful  for  preserving  plate  cultures. 
Instead  of  making  the  cultures  in  Petri's  capsules,  use  ordinary 
watch-glasses.  The  watch-glass  is  sterilised  in  a  Petri's  capsule, 
and  the  inoculated  medium  is  poured  out  into  the  watch-glass, 
allowed  to  solidify  in  the  usual  way,  and  left  in  the  Petri's  cap- 
sule until  the  colonies  of  growth  have  developed.  The  watch- 
glass  is  now  removed  from  capsule  and  a  layer  of  the  preserving 
gelatin  medium  (1),  to  which  have  been  added  a  few  drops  of 
strong  formalin,  is  allowed  to  spread  over  the  surface  of  the  culture 
medium.  When  the  layer  is  solidified  the  watch-glass  is  filled  up 
with  the  same,  and  a  clean  square  or  oblong  piece  of  glass 
(which  of  course  should  be  of  slightly  larger  diameter  than  the 
watch-glass)  is  now  carefully  placed  over  watch-glass,  care  being 
taken  that  no  air-bells  are  formed.  The  edge  of  watch-glass 
should  be  closely  applied  to  the  glass  cover  and  left  in  position 
until  the  gelatin  has  solidified.  The  superfluous  gelatin  is  now 
removed,  and  the  glasses  sealed  first  with  the  orange  shellac 
cement,  then  with  black  lacquer.  It  is  now  finished  off  by 
using  a  circular  mask  of  suitable  size. 

The  various  kinds  of  solid  media  used  in  the  cultivation  of 
bacteria,  such  as  blood  serum,  potato,  bread  paste,  etc.,  can  be 
treated  in  the  same  manner  with  excellent  results. 

General  Laboratory  Rules. — On  the  working  bench  of  every 
bacteriologist  there  should  be  a  large  dish  of  1-1000  solution  of 
mercuric  chloride  in  water.  Into  this  all  tubes,  vessels,  plates, 
hanging-drop  cultures,  etc.,  which  have  contained  bacteria  and 
with  which  he  has  finished,  ought  to  be  at  once  plunged  (in  the 
case  of  tubes  the  tube  and  plug  should  be  put  in  separately). 
On  no  account  whatever  are  such  infected  articles  to  be  left 
lying  about  the  laboratory.  The  basin  is  to  be  repeatedly 
cleaned  out.  All  the  glass  is  carefully  washed  in  repeated 
changes  of  tap  water  to  remove  the  last  trace  of  perchloride  of 
mercury,  a  very  minute  quantity  of  which  is  sufficient  to  inhibit 
growth.  Old  cultures  which  have  been  stored  for  a  time  and 
from  which  fresh  sub-cultures  have  been  made  ought  to  be 
steamed  in  the  Koch's  steriliser  for  two  or  three  hours,  or  in  the 
autoclave  for  a  shorter  period,  and  the  tubes  thoroughly  washed 
out.  Besides  a  basin  of  mercuric  chloride  solution  for  infected 
apparatus,  etc.,  there  ought  to  be  a  second  reserved  for  the 
worker's  hands  in  case  of  any  accidental  contamination.  When, 
as  in  public-health  work,  a  large  number  of  tubes  are  being 
daily  put  out  of  use,  they  may  be  placed  in  an  enamelled  slop- 
pail  and  this  when  full  is  placed  in  the  steam  steriliser. 

A  white  glazed  tile  on  which  a  bell-jar  can  be  set  is  very 


84     METHODS    OF   CULTIVATION   OF   BACTERIA 

convenient  to  have  on  a  bench.  Infective  material  in  watch- 
glasses  can  be  placed  thus  under  cover  while  investigation  is 
going  on,  and  if  anything  is  spilled  the  whole  can  be  easily 
disinfected.  In  making  examinations  of  organs  containing 
virulent  bacteria,  the  hands  should  be  previously  dipped  in 
1-1000  mercuric  chloride  and  allowed  to  remain  wet  with  this 
solution.  No  food  ought  to  be  partaken  of  in  the  laboratory, 
and  pipes,  etc.,  are  not  to  be  laid  with  their  mouth-pieces  on 
the  bench.  No  label  is  to  be  licked  with  the  tongue.  Before 
leaving  the  laboratory  the  bacteriologist  ought  to  wash  the 
hands  and  forearms  with  1-1000  mercuric  chloride  and  then 
with  yellow  soap.  In  the  case  of  any  fluid  containing  bacteria 
being  accidentally  spilt  on  the  bench  or  floor,  1-1000  mercuric 
chloride  is  to  be  at  once  poured  on  the  spot.  The  air  of  the 
laboratory  ought  to  be  kept  as  quiet  as  possible. 


CHAPTER    III. 

<• 

MICROSCOPIC  METHODS— GENERAL  BACTERIO- 
LOGICAL  DIAGNOSIS— INOCULATION  OF  ANIMALS. 

The  Microscope. — For  ordinary  bacteriological  work  a  good 
microscope  is  essential.  It  ought  to  have  a  heavy  stand,  with 
rack  and  pinion  and  fine  adjustment,  a  double  mirror  (flat  on 
one  side,  concave  on  the  other),  a  good  condenser,  with  an  iris 
diaphragm,  and  a  triple  nose-piece.  It  is  best  to  have  three 
objectives,  either  Zeiss  A,  D,  and  y^-inch  oil  immersion,  or  the 
lenses  of  other  makers  corresponding  to  these.  The  oil  immer- 
sion lens  is  essential.  It  is  well  to  have  two  eye-pieces,  say 
Nos.  2  and  4  of  Zeiss  or  lenses  of  corresponding  strengths. 
The  student  must  be  thoroughly  familiar  with  the  focussing  of 
the  light  on  the  lens  by  means  of  the  condenser,  and  also  with 
the  use  of  the  immersion  lens.  It  may  here  be  remarked  that 
when  it  is  desired  to  bring  out  in  sharp  relief  the  margins  of 
unstained  objects,  e.g.  living  bacteria  in  a  fluid,  a  narrow 
aperture  of  the  diaphragm  should  be  used,  whereas,  in  the  case 
of  stained  bacteria,  when  a  pure  colour  picture  is  desired,  the 
diaphragm  ought  to  be  widely  opened.  The  flat  side  of  the 
mirror  ought  to  be  used  along  with  the  condenser.  When  the 
observer  has  finished  for  the  time  being  with  the  immersion  lens 
he  ought  to  wipe  off  the  oil  with  a  piece  of  silk  or  very  fine 
washed  linen.  If  the  oil  has  dried  on  the  lens  it  may  be 
moistened  with  xylol — never  with  alcohol,  which  will  dissolve 
the  material  by  which  the  lens  is  fixed  in  its  metal  carrier. 

Microscopic  Examination  of  Bacteria.  1.  Hanging -drop 
Preparations. — Micro-organisms  may  be  examined  :  (1)  alive  or 
dead  in  fluids ;  (2)  in  film  preparations ;  (3)  in  sections  of 
tissues.  In  the  two  last  cases  advantage  is  always  taken  of  the 
affinity  of  bacteria  for  certain  stains.  When  they  are  to  be 
examined  in  fluids  a  drop  of  the  liquid  may  be  placed  on  a  slide 

85 


86  MICROSCOPIC   METHODS 

and  covered  with  a  cover-glass.1     It  is  more  usual,  however,  to 
employ  hanging-drop   preparations.     The  technique  of  making 
these  has  already  been  described  (p.  63).     In  examining  them 
microscopically,  it  is  necessary  to  use  a  very  small  diaphragm. 
It  is  best  to  focus  the  edge  of   the  drop  with  a   low -power 
objective,  and,  arranging  the  slide   so  that   part  of  the  edge 
crosses  the  centre  of  the  field,  to  clamp  the  preparation  in  this 
position.     A  high-power  lens  is  then  turned  into  position  and 
lowered  by  the  coarse  adjustment  to  a  short  distance  above  its 
focal  distance ;    it  is  now  carefully  screwed  down  by  the  fine 
adjustment,  the  «ye  being  kept  at  the  tube  meanwhile.     The 
shadow  of  the  edge  will  be  first  recognised,  and  then  the  bacteria 
must  be  carefully  looked  for.     Often  a  dry  lens  is  sufficient,  but 
for  some  purposes  the  oil  immersion  is  required.     If  the  bacteria 
are  small  and  motile  a  beginner  may  have  great  difficulty  in 
seeing  them,  and  it  is  well  to  practise  at  first  on  some  large  non- 
motile  form  such  as  anthrax.     In  fluid  preparations  the  natural 
appearance  of  bacteria  may  be  studied,  and  their  rate  of  growth 
determined.     The  great  use  of  such  preparations,  however,  is  to 
find  whether  or  not  the  bacteria  are  motile,  and  for  determining 
this  point  it  is  advisable  to  use  either  broth  or  agar  cultures  not 
more  than  twenty -four  hours  old.     In  the  latter  case  a   small 
fragment  of  growth  is  broken  down  in  broth  or  in  sterile  water. 
Sometimes  it  is  an  advantage  to  colour  the  solution  in  which 
the  hanging-drop  is  made  up  with  a  minute  quantity  of  an 
aniline  dye,  say  a  small  crystal  of  gentian  violet  to  100  c.c.  of 
bouillon.     Such  a  degree  of  dilution  will  not  have  any  effect  on 
the  vitality  of  the  bacteria.     Ordinarily,  living  bacteria  will  not 
take   up  a   stain,  but  even  though  they  do  not,  the  contrast 
between  the  unstained  bacteria  and  the  tinted  fluid  will  enable 
the  observer  more  easily  to  recognise  them. 

2.  Film  Preparations,  (a)  Dry  Method. — This  is  the  most 
extensively  applicable  method  of  microscopically  examining 
bacteria.  Fluids  containing  bacteria,  such  as  blood,  pus, 
scrapings  of  organs,  can  be  thus  investigated,  as  also  cultures 
in  fluid  and  solid  media.  The  first  requisite  is  a  perfectly  clean 
cover-glass.  Many  methods  are  recommended  for  obtaining 
such.  The  test  of  this  being  accomplished  is  that,  when  the 
drop  of  fluid  containing  the  bacteria  is  placed  upon  the  glass,  it 
can  be  uniformly  spread  with  the  platinum  needle  all  over  the 
surface  without  showing  any  tendency  to  retract  into  droplets. 

1  In  bacteriological  work  it  is  essential  that  cover-glasses  of  No.  1  thick- 
ness (i.e.  "14  mm.  thick)  should  be  used,  as  those  of  greater  thickness  are  not 
suitable  for  a  "i 


FILM   PREPARATIONS  87 

The  best  method  is  that  recommended  by  Van  Ermengem.  The 
cover-glasses  are  placed  for  some  time  in  a  mixture  of  con- 
centrated sulphuric  acid  6  parts,  potassium  bichromate  6  parts, 
water  100  parts,  then  washed  thoroughly  in  water  and  stored  in 
absolute  alcohol.  For  use,  a  cover-glass  is  either  dried  by 
wiping  with  a  clean  duster  or  is  simply  allowed  to  dry.  This 
method  will  amply  repay  the  trouble,  and  really  saves  time  in 
the  end.  A  clean  cover  having  been  obtained,  the  film  pre- 
paration can  now  be  made.  If  a  fluid  is  to  be  examined  a 
loopful  may  be  placed  on  the  cover -glass,  and  either  spread 
out  over  the  surface  with  the  needle,  or  another  clean  cover 
may  be  placed  on  the  top  of  the  first,  the  drop  thus  spread 
out  between  them  and  the  two  then  drawn  apart.  When 
a  culture  on  a  solid  medium  is  to  be  examined  a  loopful  of 
distilled  water  is  placed  on  the 
cover-glass  and  a  minute  par- 
ticle of  growth  rubbed  up  in  it 
and  spread  over  the  glass.  The 
great  mistake  made  by  begin- 
ners is  to  take  too  much  of  the  FlG'  4^~~C  f°r  h°ldillg 
growth.  The  point  of  the 
straight  needle  should  just  touch  the  surface  of  the  culture,  and 
when  this  is  rubbed  up  in  the  droplet  of  water  and  the  film  dried, 
there  should  be  an  opaque  cloud  just  visible  on  the  cover-glass. 
When  the  film  has  been  spread  it  must  next  be  dried  by  being 
waved  backwards  and  forwards  at  arm's-length  above  a  Bunsen 
flame.  The  film  must  then  be  fixed  on  the  glass  by  being 
passed  three  or  four  times  slowly  through  the  flame.  In  doing 
this  a  good  plan  is  to  hold  the  cover-glass  between  the  right 
forefinger  and  thumb;  if  the  fingers  just  escape  being  burned 
no  harm  will  accrue  to  the  bacteria  in  the  film. 

In  making  films  of  a  thick  fluid  such  as  pus  it  is  best  to 
spread  it  out  on  one  cover  with  the  needle.  The  result  will  be 
a  film  of  irregular  thickness,  but  sufficiently  thin  at  many  parts 
for  proper  examination.  Scrapings  of  organs  may  be  smeared 
directly  on  the  cover-glasses. 

In  the  case  of  blood,  a  fairly  large  drop  should  be  allowed  to 
spread  itself  between  two  clean  cover-glasses,  which  are  then  to 
be  slipped  apart,  and  being  held  between  the  forefinger  and 
thumb  are  to  be  dried  by  a  rapid  to-and-fro  movement  in  the 
air.  A  film  prepared  in  this  way  may  be  too  thick  at  one  edge, 
but  at  the  other  is  beautifully  thin.  If  it  is  desired  to  preserve 
the  red  blood  corpuscles  in  such  a  film  it  may  be  fixed  by  one 
of  the  following  methods  :  by  being  placed  (a)  in  a  hot-air 


88  MICROSCOPIC   METHODS 

chamber  at  120°  C.  for  half  an  hour,  (b)  in  a  mixture  of  equal 
parts  of  alcohol  and  ether  for  half  an  hour,  then  washed  and 
dried,  (c)  in  formol-alcohol  (Gulland)  (formalin  1  part,  absolute 
alcohol  9  parts)  for  five  minutes,  then  washed  and  dried,  or  (d) 
in  a  saturated  solution  of  corrosive  sublimate  for  two  or  three 
minutes,  then  washed  well  in  running  water  and  dried.  (Fig.  71 
shows  a  film  prepared  by  the  last  method.)  In  using  the 
Romanowsky  stains  no  previous  fixation  is  necessary  (vide  infra). 
In  the  case  of  urine,  the  specimen  must  be  allowed  to  stand,  and 
films  made  from  any  deposit  which  occurs;  or,  what  is  still 
better,  the  urine  is  centrifugalised,  and  films  made  from  the 
deposit  which  forms.  After  dried  films  are  thus  made  from 
urine  it  is  an  advantage  to  place  a  drop  of  distilled  water  on  the 
film  and  heat  gently  to  dissolve  the  deposit  of  salts  ;  then  wash 
in  water  and  dry.  In  this  way  a  much  clearer  picture  is 
obtained  when  the  preparation  is  stained. 

Within  recent  years  it  has  become  common  to  make  blood 
films  on  ordinary  microscopic  slides  instead  of  upon  cover- 
glasses.  Here  the  slides  must  be  clean.  This  can  be  effected  by 
washing  thoroughly  first  with  weak  alkali  and  then  with  water 
and  storing  in  alcohol.  For  use,  a  slide  is  taken  from  the 
alcohol  and  the  fluid  adhering  to  it  set  on  fire  and  allowed  to 
burn  off,  a  dry  clean  slide  being  thus  obtained.  To  make  a  film 
on  such,  a  small  drop  of  blood  is  placed  near  one  end,  the  edge 
of  a  second  clean  slide  is  lowered  through  the  drop  on  to  the 
surface  of  the  glass  on  which  the  blood  has  been  placed.  This 
second  slide  is  held  at  an  angle  to  the  first,  on  which  it  rests  by 
its  edge.  The  droplet  of  blood  by  capillarity  spreads  itself  in 
the  angle  between  the  two  slides.  The  edge  of  the  second  slide  is 
then  stroked  along  the  surface  of  the  first  slide,  and  in  this 
procedure  the  blood  is  spread  out  in  a  film  whose  thickness  can 
be  regulated  by  the  angle  formed  by  the  second  slide.  Large- 
sized  films  can  thus  be  obtained,  and  when  these  are  stained  they 
are  often  examined  without  any  cover-glass  being  placed  upon 
them.  A  drop  of  cedar  oil  is  placed  on  the  preparation,  and 
after  use  this  can  be  removed  by  the  careful  application  of 
xylol. 

Films  dried  and  fixed  by  the  above  methods  are  now  ready  to 
be  stained  by  the  methods  to  be  described  below. 

(6)  Wet  Method. — If  it  is  desired  to  examine  the  fine 
histological  structure  of  the  cells  of  a  discharge  as  well  as  to 
investigate  the  bacteria  present,  it  is  advisable  to  substitute 
"  wet "  films  for  the  "  dried  "  films,  the  preparation  of  which  has 
been  described.  The  nuclear  structure,  mitotic  figures,  etc.,  are 


EXAMINATION   OF   BACTERIA  IN   TISSUES      89 

by  this  method  well  preserved,  whereas  these  are  considerably 
distorted  in  dried  films.  The  initial  stages  in  the  preparation  of 
wet  films  are  the  same  as  above,  but  instead  of  being  dried  in 
air  they  are  placed,  while  still  wet,  film  downwards  in  the 
fixative.  The  following  are  some  of  the  best  fixing  methods  : — 

(a)  A  saturated  solution  of  perchloride  of  mercury  in  75  per  cent 
sodium  chloride  ;  fix  for  five  minutes.  Then  place  the  films  for  half  an 
hour,  with  occasional  gentle  shaking,  in  75  per  cent  sodium  chloride 
solution  to  wash  out  the  corrosive  sublimate  ;  they  are  thereafter  washed 
in  successive  strengths  of  methylated  spirit.  After  this  treatment  the 
films  are  stained  and  treated  as  if  they  were  sections. 

(&)  Formol-alcohol — formalin  1  part,  absolute  alcohol  9.  Fix  films 
for  three  minutes  ;  then  wash  well  in  methylated  spirit.  This  is  an 
excellent  and  very  rapid  method. 

(c)  Another  excellent  method  of  fixing  has  been  devised  by  Gulland. 
The  fixing  solution  has  the  composition — absolute  alcohol  25  c.c.,  pure 
ether  25  c.c.,  alcoholic  solution  of  corrosive  sublimate  (2  grm.  in  10  c.c. 
of  alcohol)  about  5  drops.  The  films  are  placed  in  this  solution  for  five 
minutes  or  longer.  They  are  then  washed  well  in  water,  and  are  ready 
for  staining.  A  contrast  stain  can  be  applied  at  the  same  time  as  the 
fixing  solution,  by  saturating  the  25  c.c.  of  alcohol  with  eosin  before 
mixing.  Thereafter  the  bacteria,  etc.,  may  be  stained  with  methylene- 
blue  or  other  stain,  as  described  below.  This  method  has  the  advantage 
over  (a)  that,  as  a  small  amount  of  corrosive  sublimate  is  used,  less 
washing  is  necessary  to  remove  it  from  the  preparation,  and  deposits  are 
less  liable  to  occur. 

3.  Examination  of  Bacteria  in  Tissues. — For  the  examina- 
tion of  bacteria  in  the  tissues,  the  latter  must  be  fixed  and 
hardened,  in  preparation  for  being  cut  with  a  microtome. 
Fixation  consists  in  so  treating  a  tissue  that  it  shall  permanently 
maintain,  as  far  as  possible,  the  condition  it  was  in  when  re- 
moved from  the  body.  Hardening  consists  in  giving  such  a 
fixed  tissue  sufficient  consistence  to  enable  a  thin  section  of  it 
to  be  cut.  A  tissue,  after  being  hardened,  may  be  cut  in  a 
freezing  microtome  (e.g.  Cathcart's  or  one  of  the  newer  instru- 
ments in  which  the  freezing  is  accomplished  by  compressed 
carbonic  acid  gas),  but  far  finer  results  can  be  obtained  by 
embedding  the  tissue  in  solid  paraffin  and  cutting  with  some  of 
the  more  delicate  microtomes  of  which,  for  pathological  purposes, 
the  small  Cambridge  rocker  is  by  far  the  best.  For  bacterio- 
logical purposes  embedding  in  celloidin  is  not  advisable,  as  the 
celloidin  takes  on  the  aniline  dyes  which  are  used  for  staining 
bacteria,  and  is  apt  thus  to  spoil  the  preparation,  and  besides 
thinner  sections  can  be  obtained  by  the  paraffin  method. 

The  Fixation  and  Hardening  of  Tissues. — The  following 
are  amongst  the  best  methods  for  bacteriological  purposes  : — 

(a)  Absolute  alcohol  may  be  used  for  the  double  purpose  of  fixing  and 
hardening.  If  the  piece  of  tissue  is  not  more  than  t?  inch  in  thickness  it 


90  MICROSCOPIC   METHODS 

is  sufficient  to  keep  it  in  this  reagent  for  a  few  hours.  If  the  pieces 
are  thicker  a  longer  exposure  is  necessary,  and  in  such  cases  it  is  better 
to  change  the  alcohol  at  the  end  of  the  first  twenty-four  hours.  The 
tissue  must  be  tough  without  being  hard,  and  the  necessary  consistence, 
as  estimated  by  feeling  with  the  lingers,  can  only  be  judged  of  after 
some  experience.  If  the  tissues  are  not  to  be  cut  at  once,  they  may  be 
preserved  in  50  per  cent  spirit. 

(b)  Formal- alcohol — formalin  1,  absolute  alcohol  9.     Fix  for  not  more 
than  twenty-four  hours  ;  then  place  in  absolute  alcohol  if  the  tissue  is 
to  be  embedded  at  once,  in  50  per  cent  spirit  if  it  is  to  be  kept  for  some 
time.     For  small  pieces  of  tissue  fixation  for  twelve  hours  or  even  less  is 
sufficient.     The  method  is  a  rapid  and  very  satisfactory  one. 

(c)  Corrosive  sublimate  is  an  excellent  fixing  agent.     It  is  best  used 
as  a  saturated  solution  in  '75  per  cent  sodium  chloride  solution.     Dis- 
solve  the   sublimate   in   the   salt   solution  by  heat ;  the  separation  of 
crystals  on  cooling  shows  that   the  solution   is   saturated.     For   small 
pieces  of  tissue  £  inch  in  thickness,  twelve  hours'  immersion  is  sufficient. 
If  the  pieces  are  larger,  twenty-four  hours  is  necessary.     They  should 
then  be  tied  up  in  a  piece  of  gauze,  and  placed' in  a  stream  of  running 
water  for  from  twelve  to  twenty-four  hours,  according  to  the  size  of  the 
pieces,  to  wash  out  the  excess  of  sublimate.     They  are  then  placed  for 
twenty-four   hours   in  each  of  the  following  strengths   of  methylated 
spirit  (free  from  naphtha1) :  30  per  cent,  60  per  cent,  and  90  per  cent. 
Finally  they  are  placed  in  absolute  alcohol  for  twenty-four  hours  and 
are  then  ready  to  be  prepared  for  cutting. 

If  the  tissue  is  very  small,  as  in  the  case  of  minute  pieces  removed 
for  diagnosis,  the  stages  may  be  all  compressed  into  twenty-four  hours. 
In  fact  after  fixation  in  corrosive  the  tissue  may  be  transferred  directly 
to  absolute  alcohol,  the  perchloride  of  mercury  being  removed  after  the 
sections  are  cut,  as  will  be  afterwards  described. 

(d)  Methylated   Spirit. — Small    pieces   of    tissue   may   be    placed-  in 
methylated  spirit,  which  is  to  be  changed  after  the  first  day.     In  from 
six  to  seven  days  they  will  be  hardened.     If  the  pieces  are   large,  a 
longer  time  is  necessary. 

The  Cutting  of  Sections. — 1.  By  Means  of  the  Freezing 
Microtome. — Pieces  of  tissue  hardened  by  any  of  the  above 
methods  must  have  all  the»alcohol  removed  from  them  by  wash- 
ing in  running  water  for  twenty-four  hours.  They  are  then 
placed  for  from  twelve  to  twenty-four  hours  (according  to  their 
size)  in  a  thick  syrupy  solution  containing  two  parts  of  gum 
arabic  and  one  part  of  sugar.  They  are  then  cut  on  a  freezing 
microtome  and  placed  for  a  few  hours  in  a  bowl  of  water  so  that 
the  gum  and  syrup  may  dissolve  out.  They  are  then  stained  or 
they  may  be  stored  in  methylated  spirit. 

1  In  Britain  ordinary  commercial  methylated  spirit  has  wood  naphtha 
added  to  it  to  discourage  its  being  used  as  a  beverage.  The  naphtha  being 
insoluble  in  water  a  milky  fluid  results  from  the  dilution  of  the  spirit.  By 
law,  chemists  can  only  sell  8  ounces  of  pure  spirit  at  a  time.  Most  patho- 
logical laboratories  are,  however,  licensed  by  the  Excise  to  buy  "industrial 
spirit, "  which  contains  only  one-nineteenth  of  wood  naphtha. 


THE   CUTTING   OF   SECTIONS  91 

2.  Embedding  and  Cutting  in  Solid  Paraffin. — This  method 
gives  by  far  the  finest  results^nd  should  always  be  adopted 
when  practicable.  The  principle  is  the  impregnation  of  the 
tissue  with  paraffin  in  the  melted  state.  This  paraffin  when  it 
solidifies  gives  support  to  all  the  tissue  elements.  The  method 
involves  that,  after  hardening,  the  tissue  shall  be  thoroughly 
dehydrated,  and  then  thoroughly  permeated  by  some  solvent 
of  paraffin  which  will  expel  the  dehydrating  fluid  and  prepare 
for  the  entrance  of  the  paraffin.  The  solvents  most  in  use  are 
chloroform,  cedar  oil,  xylol,  and  turpentine ;  of  these  chloroform 
and  cedar  oil  are  the  best,  the  former  being  preferred  as  it  per- 
meates the  tissue  more  rapidly.  The  more  gradually  the  tissues 
are  changed  from  reagent  to  reagent  in  the  processes  to  be  gone 
through,  the  more  successful  is  the  result.  A  necessity  of  the 
process  is  an  oven  with  hot-water  jacket,  in  which  the  paraffin 
can  be  kept  at  a  constant  temperature  just  above  its  melting- 
point,  a  gas  regulator,  e.g.  Eeichert's,  being  of  course  necessary. 
The  tissues  occurring  in  pathological  work  have  a  tendency  to 
become  brittle  if  overheated,  and  therefore  the  best  results  are 
not  obtained  by  using  paraffin  melting  about  58°  C.,  such  as  is 
employed  in  most  biological  laboratories.  We  have  used  for 
some  years  a  mixture  of  one  part  of  paraffin,  melting  at  48°,  and 
two  parts  of  paraffin  melting  at  54°  C.  This  mixture  has  a 
melting-point  between  52°  and  53°  C.,  and  it  serves  all  ordinary 
purposes  well.  An  excellent  quality  of  paraffin  is  that  known 
as  the  "Cambridge  paraffin,"  but  many  scientific-instrument 
makers  supply  paraffins  which,  for  ordinary  purposes,  are  quite 
as  good,  and  much  cheaper.  The  successive  steps  in  the  process 
of  paraffin  embedding  are  as  follows  : — 

1.  Pieces  of  tissue,  however  hardened,  are  placed  in  fresh  absolute 
alcohol  for  twenty-four  hours  in  order  to  their  complete  dehydration. 

2.  Transfer  now  to  a  mixture  of  equal  parts  of  absolute  alcohol  and 
chloroform  for  twenty- four  hours. 

3.  Transfer  to  pure  chloroform  for  twenty- four  hours  or  longer.     At 
the  end  of  this  time  the  tissues  should  sink  or  float  heavily. 

4.  Transfer  now  to  a  mixture  of  equal  parts  of  chloroform  and  paraffin 
and  place  on  the  top  of  the  oven  for  from  twelve  to  twenty-four  hours. 
If  the  temperature  there  is  not  sufficient  to  keep  the  mixture  melted 
then  they  must  be  put  inside. 

5.  Place  in  pure  melted  paraffin  in  the  oven  for  twenty-four  hours. 
For  holding  the  paraffin  containing  the  tissues,  small  tin  dishes  such  as  are 
used   by  pastry-cooks  will  be   found  very  suitable.     There  must   be  a 
considerable  excess  of  paraffin  over  the  bulk  of  tissue  present,  otherwise 
sufficient  chloroform  will  be  present  to  vitiate  the  final  result  and  not 
give  the  perfectly  hard  block  obtained  with  pure  paraffin.     With  ex- 
perience,  the   persistence  of  the   slightest  trace  of  chloroform  can   be 
recognised  by  smell. 


92  MICROSCOPIC   METHODS 

In  the  case  of  very  small  pieces  of  tissue  the  time  given  for  each  stage 
may  be  much  shortened,  and  where  haste  is  desirable  Nos.  2  and  4  may 
be  omitted.  Otherwise  it  is  better  to  carry  out  the  process  as  de- 
scribed. When  it  is  advisable  to  avoid  all  shrinkage  it  is  well  to  change 
the  paraffin  every  few  hours  during  the  embedding  process. 

6.  Cast  the  tissues  in  blocks  of  paraffin  as  follows  :  Pairs  of  L-shaped 
pieces  of  metal  made  for  the  purpose  by  instrument  makers  must  be  at 
hand.  By  laying  two  of  these  together  on  a  glass  plate,  a  rectangular 
trough  is  formed.  This  is  filled  with  melted  paraffin  taken  from  a  stock 
in  a  separate  dish.  In  it  is  immersed  the  piece  of  tissue,  which  is  lifted 
out  of  its  pure  paraffin  bath  with  heated  forceps.  The  direction  in 
which  it  is  to  be  cut  must  be  noted  before  the  paraffin  becomes  opaque. 
When  the  paraffin  has  begun  to  set,  the  glass  plate  and  trough  have 
cold  water  run  over  them.  When  the  block  is  cold,  the  metal  L's  are 
broken  off,  and,  its  edges  having  been  pared,  it  is  stored  in  a  pill-box. 

The  Cutting  of  Paraffin  Sections. — Sections  must  be  cut  as 
thin  as  possible,  the  Cambridge  rocking  microtome  being,  on 
the  whole,  most  suitable.  They  should  not  exceed  8  //,  in  thick- 


FIG.  43. — Needle  with  square  of  paper  on  end  for  manipulating  paraffin 
sections. 

ness,  and  ought,  if  possible,  to  be  about  4  //..  For  their  mani- 
pulation it  is  best  to  have  two  needles  on  handles,  two  camel's- 
hair  brushes  on  handles,  and  a  needle  with  a  rectangle  of  stiff 
writing  paper  fixed  on  it  as  in  the  diagram  (Fig.  43).  When 
cut,  sections  are  floated  on  the  surface  of  a  beaker  of  water  kept 
at  a  temperature  about  10°  C.  below  the  melting-point  of  the 
paraffin.  On  the  surface  of  the  warm  water  they  become  perfectly 
flat. 

Fixation  on  Ordinary  Slides,  (a]  Gulland's  Method. — A  supply  of 
slides  well  cleaned  being  at  hand,  one  of  them  is  thrust  obliquely  into 
the  water  below  the  section,  a  corner  of  the  section  is  fixed  on  it  with  a 
needle  and  the  slide  withdrawn.  The  surplus  of  water  being  wiped  off 
with  a  cloth,  the  slide  is  placed  on  a  support,  with  the  section  down- 
wards, and  allowed  to  remain  on  the  top  of  the  paraffin  oven  or  in  a 
bacteriological  incubator  for  from  twelve  to  twenty-four  hours.  It  will 
then  be  sufficiently  fixed  on  the  slide  to  withstand  all  the  manipulations 
necessary  during  staining  and  mounting. 

(b)  Fixation  by  Manns  Method. — This  has  the  advantage  of  being 
more  rapid  than  the  previous  one.  A  solution  of  albumin  is  prepared 
by  mixing  the  white  of  a  fresh  egg  with  ten  parts  of  distilled  water  and 
filtering.  Slides  are  made  perfectly  clean  with  alcohol.  One  is  dipped 
into  the  solution  and  its  edge  is  then  drawn  over  one  surface  of  another 
slide  so  as  to  leave  on  it  a  thin  film  of  albumin.  This  is  repeated  with 
the  others.  As  each  is  thus  coated,  it  is  leant,  with  the  film  down- 


AND   CLEARING  93 

wards,  on  a  ledge  till  dry,  and  then  the  slides  are  stored  in  a  wide 
stoppered  jar  till  needed.  The  floating  out  is  performed  as  before. 
The  albuminised  side  of  the  slide  is  easily  recognised  by  the  fact  that  if 
it  is  breathed  on,  the  breath  does  not  condense  on  it.  The  great 
advantage  of  this  method  is  that  the  section  is  fixed  after  twenty  to 
thirty  minutes'  drying  at  37°  C.  If  the  tissue  has  been  hardened  in  any 
of  the  bichromate  solutions  and  embedded  in  paraffin,  this  or  some 
corresponding  method  of  fixing  the  sections  on  the  slide  must  be  used. 

Preparation  of  Paraffin  Sections  for  Staining. — Before  stain- 
ing, the  paraffin  must  be  removed  from  the  section.  This  is 
best  done  by  dropping  on  xylol  out  of  a  drop  bottle.  When  the 
paraffin  is  dissolved  out,  the  superfluous  xylol  is  wiped  off  with 
a  cloth  and  a  little  absolute  alcohol  dropped  on.  When  the 
xylol  is  removed  the  superfluous  alcohol  is  wiped  off  and  a 
little  50  per  cent  methylated  spirit  dropped  on.  During  these 
procedures  sections  must  on  no  account  be  allowed  to  dry. 
The  sections  are  now  ready  to  be  stained.  Deposits  of  crystals 
of  corrosive  sublimate  often  occur  in  sections  which  have  been 
fixed  by  this  reagent.  These  can  be  removed  by  placing  the 
sections,  before  staining,  for  a  few  minutes  in  equal  parts  of 
Gram's  iodine  solution  (p.  99)  and  water,  and  then  washing  out 
the  iodine  with  methylated  spirit. 

To  save  repetition  we  shall  in  treating  of  stains  suppose  that, 
with  paraffin  sections,  the  above  preliminary  steps  have  already 
been  taken,  and  further  that  sections  cut  by  a  freezing  microtome 
are  also  in  spirit  and  water. 

Dehydration  and  Clearing. — It  is  convenient,  first  of  all,  to 
indicate  the  final  steps  to  be  taken  after  a  specimen  is  stained. 
Dry  films  after  being  stained  are  washed  in  water,  dried  and 
mounted  in  xylol  balsam ;  wet  films  and  sections  must  be 
dehydrated,  cleared,  and  then  mounted  in  xylol  balsam. 

Dehydration  is  most  commonly  effected  with  absolute  alcohol. 
Alcohol,  however,  sometimes  decolorises  the  stained  organisms 
more  than  is  desirable,  and  therefore  Weigert  devised  the 
following  method  of  dehydrating  and  clearing  by  aniline  oil, 
which,  though  it  may  decolorise  somewhat,  does  not  do  so  to  the 
same  extent  as  alcohol.  As  much  as  possible  of  the  water  being 
removed,  the  section  placed  on  a  slide  is  partially  dried  by 
draining  with  fine  blotting-paper.  Some  aniline  oil  is  placed  on 
the  section  and  the  slide  moved  to  and  fro.  The  section  is 
dehydrated  and  becomes  clear.  The  process  may  be  accelerated 
by  heating  gently.  The  preparation  is  then  treated  with  a 
mixture  of  two  parts  of  aniline  oil  and  one  part  of  xylol,  and 
then  with  xylol  alone,  after  which  it  is  mounted  in  xylol  balsam. 
Balsam  as  ordinarily  supplied  has  often  an  acid  reaction,  and 


94  MICEOSCOPIC   METHODS 

preparations  stained  with  aniline  dyes  are  apt  to  fade  when 
mounted  in  it.  It  is  accordingly  a  great  advantage  to  use  the 
acid-free  balsam  supplied  by  Griibler.  Paraffin  sections  can 
usually  be  dehydrated  and  cleared  by  the  mixture  of  aniline  oil 
and  xylol  alone. 

Sections  stained  for  bacteria  should  always  be  cleared,  at 
least  finally,  in  xylol,  for  the  same  reason  that  xylol  balsam  is 
to  be  used  for  mounting  films,  viz.  that  it  dissolves  out  aniline 
dyes  less  readily  than  such  clearing  reagents  as  clove  oil,  etc. 
Xylol,  however,  requires  the  previous  dehydration  to  have  been 
more  complete  than  clove  oil,  which  will  clear  a  section  readily 
when  the  dehydration  has  been  only  partially  effected  by,  say, 
methylated  spirit.  If  a  little  decolorisation  of  a  section  is  still 
required  before  mounting,  clove  oil  may  be  used  to  commence 
the  clearing,  the  process  being  finished  with  xylol.  With  a  little 
experience  the  progress,  not  only  of  these  processes  but  also  of 
staining,  can  be  very  accurately  judged  of  by  observing  the 
appearances  under  a  low  objective. 


THE  STAINING  OF  BACTERIA. 

Staining  Principles. — To  speak  generally,  the  protoplasm  of 
bacteria  reacts  to  stains  in  a  manner  similar  to  the  nuclear 
chromatin,  though  sometimes  more  and  sometimes  less  actively. 
The  bacterial  stains  par  excellence  are  the  basic  aniline  dyes. 
These  dyes  are  more  or  less  complicated  compounds  derived 
from  the  coal-tar  product  aniline  (C0H5 .  NH9).  Many  of  them 
have  the  constitution  of  salts.  Such  compounds  are  divided 
into  two  groups  according  as  the  staining  action  depends  on  the 
basic  or  the  acid  portion  of  the  molecule.  Thus  the  acetate  of 
rosaniline  derives  its  staining  action  from  the  rosaniline.  It  is 
therefore  called  a  basic  aniline  dye.  On  the  other  hand, 
ammonium  picrate  owes  its  action  to  the  picric  acid  part  of  the 
molecule.  It  is  therefore  termed  an  acid  aniline  dye.  These 
two  groups  have  affinities  for  different  parts  of  the  animal  cell. 
The  basic  stains  have  a  special  affinity  for  the  nuclear  chromatin, 
the  acid  for  the  protoplasm  and  various  formed  elements.  Thus 
it  is  that  the  former — the  basic  aniline  dyes — are  especially  the 
bacterial  stains. 

The  number  of  basic  aniline  stains  is  very  large.  The  following  are 
the  most  commonly  used  : — 

Violet  Stains. — Methyl- violet,  R-5R  (synonyms  :  Hoffmann's  violet, 
dahlia). 

Gentian-violet  (synonyms  :  benzyl-violet,  Pyoktanin). 


THE   STAINING   OF   BACTERIA  95 

Crystal  violet. 

Blue  Stains. — Methylene-blue1  (synonym  :  phenylcne-blue). 
Victoria-blue. 
Thionin-blue. 

Red  Stains. — Basic  fuclisin  (synonyms  :  basic  rubin,  magenta). 
Safranin  (synonyms  :  fuchsia,  Girofle). 

Brown  Stain. — Bismarck  -  brown  (synonyms:  vesuvin,  phenylene- 
brown). 

It  is  of  the  greatest  importance  that  the  stains  used  by  the 
bacteriologist  should  be  good,  and  therefore  it  is  advisable  to 
obtain  those  prepared  by  Griibler  of  Leipzig.  One  is  then 
perfectly  sure  that  one  has  got  the  right  stain. 

Of  the  stains  specified,  the  violets  and  reds  are  the  most 
intense  in  action,  especially  the  former.  It  is  thus  easy  in  using 
them  to  overstain  a  specimen.  Of  the  blues,  methylene-blue 
probably  gives  the  best  differentiation  of  structure,  and  it  is 
difficult  to  overstain  with  it.  Thionin-blue  also  gives  good 
differentiation  and  does  not  readily  overstain.  Its  tone  is  deeper 
than  that  of  methylene-blue  and  it  approaches  the  violets  in  tint. 
Bismarck-brown  is  a  weak  stain,  but  is  useful  for  some  purposes. 
Formerly  it  was  much  used  in  photomicrographic  work,  as  it 
was  less  actinic  than  the  other  stains.  It  is  not,  however, 
needed  now,  on  account  of  the  improved  sensitiveness  of  plates. 

It  is  most  convenient  to  keep  saturated  alcoholic  solutions 
of  the  stains  made  up,  and  for  use  to  filter  a  little  into  about 
ten  times  its  bulk  of  distilled  water  in  a  watch-glass.  A  solution 
of  good  body  is  thus  obtained.  Most  bacteria  (except  those  of 
tubercle,  leprosy,  and  a  few  others)  will  stain  in  a  short  time  in 
such  a  fluid.  Watery  solutions  may  also  be  made  up,  e.g.  a 
saturated  watery  solution  of  methylene-blue  or  a  1  per  cent 
solution  of  gentian- violet.  Stains  must  always  be  filtered  before 
use ;  otherwise  there  may  be  deposited  on  the  preparation 
granules  which  it-  is  impossible  to  wash  off.  The  violet  stains  in 
solution  in  water  have  a  great  tendency  to  decompose.  Only 
small  quantities  should  therefore  be  prepared  at  a  time. 

The  Staining  of  Cover-glass  Films. — Films  are  made  from 
cultures  as  described  above.  The  cover-glass  may  be  floated  on 
the  surface  of  the  stain  in  a  watch-glass,  or  the  cover-glass  held  in 
forceps  with  film  side  uppermost  may  have  as  much  stain  poured 
on  it  as  it  will  hold.  When  the  preparation  has  been  exposed  for 
the  requisite  time,  usually  a  few  minutes,  it  is  well  washed  in 
tap  water  in  a  bowl,  or  with  distilled  water  with  such  a  simple 
contrivance  as  that  figured  (Fig.  44).  The  figure  explains  itself. 

1  This  is  to  be  distinguished  from  methyl-blue,  which  is  a  different  com- 
pound. 


96 


MICROSCOPIC   METHODS 


When  the  film  has  been  washed  the  surplus  of  water  is  drawn 
off  with  a  piece  of  filter-paper,  the  preparation  is  carefully  dried 
high  over  a  flame,  a  drop  of  xylol  balsam  is  applied,  and  the 
cover-glass  mounted  on  a  slide.  It  is  sometimes  advantageous 
to  examine  films  in  a  drop  of  water  in  place  of  balsam.  The 
films  can  be  subsequently  dried  and  mounted  permanently.  In 
the  case  of  tubercle,  special  stains  are  necessary  (p.  100),  but  with 
this  exception,  practically  all  bacterial  films  made  from  cultures 
can  be  stained  in  this  way.  Some  bac- 
teria, e.g.  typhoid,  glanders,  take  up  the 
stains  rather  slowly,  and  for  these  the 
more  intensive  stains,  red  or  violet,  are 
to  be  preferred. 

Films  of  fluids  from  the  body  (blood, 
pus,  etc.)  can  be  generally  stained  in  the 
same  way,  and  this  is  often  quite  suffi- 
cient for  diagnostic  purposes.  The  blue 
dyes  are  here  preferable,  as  they  do  not 
readily  overstain.  In  the  case  of  such 
fluids,  if  the  histological  elements  also 
claim  attention  it  is  best  first  to  stain 
the  cellular  protoplasm  with  a  one  to 
two  per  cent  watery  solution  of  eosin 
(which  is  an  acid  dye),  and  then  to  use 
a  blue  which  will  stain  the  bacteria  and 
the  nuclei  of  the  cells.  The  Roman  owsky 
stains  (v.  p.  105)  are  here  most  useful,  as 
by  these  the  preparations  are  fixed  as 
well  as  stained.  Fixation  by  heat  which 
is  apt  to  injure  delicate  cellular  structures 
is  thus  avoided.  In  the  case  of  films 
made  from  urine,  where  there  is  little 
or  no  albuminous  matter  present,  the 
bacteria  may  be  imperfectly  fixed  on 

the  slide,  and  are  thus  apt  to  be  washed  off.  In  such  a  case  it 
is  well  to  modify  the  staining  method.  A  drop  of  stain  is 
placed  on  a  slide,  and  the  cover-glass,  film-side  down,  lowered 
upon  it.  After  the  lapse  of  the  time  necessary  for  staining, 
a  drop  of  water  is  placed  at  one  side  of  the  cover-glass  and  a 
little  piece  of  filter-paper  at  the  other  side.  The  result  is  that 
the  stain  is  sucked  out  by  the  filter-paper.  By  adding  fresh 
drops  of  water  and  using  fresh  pieces  of  filter-paper,  the  speci- 
men is  washed  without  any  violent  application  of  water,  and 
the  bacteria  are  not  displaced. 


FIG.  44.  —  Syphon  wash- 
bottle  for  distilled  water 
used  in  washing  prepara- 
tions. 


MORDANTS   AND   DECOLORISING  AGENTS      97 

For  the  general  staining  of  films  a  saturated  watery  solution 
of  methylene-blue  will  be  found  to  be  the  best  stain  to  com- 
mence with,  the  Gram  method  (v.  infra)  is  then  applied,  and 
subsequently  any  special  stains  which  may  appear  advisable. 

The  Use  of  Mordants  and  Decolorising  Agents. — In  films  of 
blood  and  pus,  and  still  more  so  in  sections  of  tissues,  if  the  above 
methods  are  used,  the  tissue  elements  may  be  stained  to  such  an 
extent  as  to  quite  obscure  the  bacteria.  Hence  many  methods 
have  been  devised  in  which  the  general  principle  may  be  said  to 
be  (a)  the  use  of  substances  which,  while  increasing  the  staining 
power,  tend  to  fix  the  stain  in  the  bacteria,  and  (b)  the  subse- 
quent treatment  by  substances  which  decolorise  the  overstained 
tissues  to  a  greater  or  less  extent,  while  they  leave  the  bacteria 
coloured.  The  staining  capacity  of  a  solution  may  be  increased — 

(a)  By  the  addition  of  substances  such  as  carbolic  acid, 
aniline  oil,  or  metallic  salts. 

(6)  By  the  addition  of  alkalies,  such  as  caustic  potash  or 
ammonium  carbonate,  in  weak  solution. 

(c)  By  the  employment  of  heat. 

(d)  By  long  duration  of  the  staining  process. 

As  decolorising  agents  we  use  chiefly  mineral  acids  (hydro- 
chloric, nitric,  sulphuric),  vegetable  acids  (especially  acetic  acid), 
alcohol  (either  methylated  spirit  or  absolute  alcohol),  or  a  com- 
bination of  spirit  and  acid,  e.g.  methylated  spirit  with  a  drop  or 
two  of  hydrochloric  acid  added,  also  various  oils,  e.g.  aniline, 
clove,  etc.  In  most  cases  about  thirty  drops  of  acetic  acid  in  a 
bowl  of  water  will  be  sufficient  to  remove  the  excess  of  stain 
from  over-stained  films  and  sections.  More  of  the  acid  may,  of 
course,  be  added  if  necessary. 

Hot  water  also  decolorises  to  a  certain  extent ;  over-stained 
films  can  be  readily  decolorised  by  placing  a  drop  of  water  on 
the  film  and  heating  gently  over  a  flame. 

When  preparations  have  been  sufficiently  decolorised  by  an 
acid,  they  should  be  well  washed  in  tap  water,  or  in  distilled 
water  with  a  little  lithium  carbonate  added. 

The  methods  embracing  the  use  of  a  stain  with  a  mordant, 
and  a  decoloriser,  are  very  numerous,  and  we  can  only  enumerate 
the  best  of  them. 

Different  organisms  take  up  and  retain  the  stains  with 
various  degrees  of  intensity,  and  thus  duration  of  staining  and 
decolorising  must  be  modified  accordingly.  We  sometimes  have 
to  deal  with  bacteria  which  show  a  special  tendency  to  be 
decolorised.  This  tendency  can  be  obviated  by  adding  a  little 


98  MICROSCOPIC   METHODS 

of  the  stain  to  the  alcohol,  or  aniline  oil,  employed  in  dehydra- 
tion. In  the  latter  case  a  little  of  the  stain  is  rubbed  down  in 
the  oil.  The  mixture  is  allowed  to  stand.  After  a  little  time  a 
clear  layer  forms  on  the  top  with  stain  in  solution,  and  this  can 
be  drawn  off  with  a  pipette. 

When  methylene-blue,  methyl- violet,  or  gentian-violet  is  used, 
the  stain  can,  after  the  proper  degree  of  decolorisation  has  been 
reached,  be  fixed  in  the  tissues  by  treating  for  a  minute  with 
ammonium  molybdate  (2|  per  cent  in  water). 

The  Formulae  of  some  of  the  more  commonly  used  Stain  Combinations. 

1.  Loffler's  Methylene-blue. 

Saturated  solution  of  methylene-blae  in  alcohol  .  .        30  c.c. 

Solution  of  potassium  hydrate  in  distilled  water  (1-10,000)      .     100   ,, 

(This  dilute  solution  may  be  conveniently  made  by  adding  1  c.c.  of  a 
1  per  cent  solution  to  99  c.c.  of  water.) 

Sections  may  be  stained  in  this  mixture  for  from  a  quarter  of  an  hour 
to  several  hours.  They  do  not  readily  overstain.  The  tissue  containing 
the  bacteria  is  then  decolorised  if  necessary  with  ^-1  per  cent  acetic  acid, 
till  it  is  a  pale  blue-green.  The  section  is  washed  in  water,  rapidly 
dehydrated  with  alcohol  or  aniline  oil,  cleared  in  xylol,  and  mounted. 

The  tissue  may  be  contrast  stained  with  eosin.  If  this  is  desired, 
after  decolorisation  wash  Avith  water,  place  for  a  few  seconds  in  1  per  cent 
solution  of  eosin  in  absolute  alcohol,  rapidly  complete  dehydration  with 
pure  absolute  alcohol,  and  proceed  as  before. 

Films  may  be  stained  with  Loffler's  blue  by  five  minutes'  exposure  or 
longer  in  the  cold.  They  usually  do  not  require  decolorisation,  as  the 
tissue  elements  are  not  overstained. 

2.  Kuhne's  Methylene-blue. 

Methylene-blue        .         .         .  1  '5  gr. 

Absolute  alcohol      .         .         .         .     10  c.c. 
Carbolic  acid  solution  (1-20)     .         .   100   ,, 

Stain  and  decolorise  as  with  Loffler's  blue,  or  decolorise  with  very 
weak  hydrochloric  acid  (a  few  drops  in  a  bowl  of  water). 

3.  Carbol-Thionin-blue.-M.ake  up  a  stock  solution  consisting  of  1 
gramme  of  thionin-blue  dissolved  in  100  c.c.  carbolic  acid  solution  (1-40). 
For  use,  dilute  1  volume  with  3  of  water  and  filter.     Stain  sections  for 
five  minutes  or  upwards.     Wash  very  thoroughly  with  water,  otherwise 
a  deposit  of  crystals  may  occur  in  the  subsequent  stages.     Decolorise 
with  very  weak  acetic  acid.     A  few  drops  of  the  acid  added  to  a  bowl 
of  water   are   quite   sufficient.      Wash   again   thoroughly  with   water. 
Dehydrate   with  absolute  alcohol.      Thionin-blue   stains  more   deeply 
than  methylene-blue,  and  gives  equally  good  differentiation.     It  is  very 
suitable  for  staining  typhoid  and  glanders  bacilli  in  sections.     Cover- 
glass    preparations    stained    by   this   method    do    not   usually   require 
decolorisation.     As  a  contrast  stain,  1  per  cent  watery  solution  of  eosin 
may  be  used  before  staining  with  the  thionin. 

4.  Gentian-violet  in  Aniline  Oil  Water. — Two  solutions  have  here  to 
be  made  up.     (a)  Aniline  oil  water.      Add  about  5  c.c.  aniline  oil  to 
100   c.c.   distilled  water  in  a  flask,  and  shake  violently  till  as  much  as 
possible  of  the  oil  has  dissolved.     Filter  and  keep  in  a  covered  bottle 


GRAM'S   STAIN  99 

to  prevent  access  of  light,  (b)  Make  a  saturated  solution  of  gentian- 
violet  in  alcohol.  When  the  stain  is  to  be  used,  1  part  of  (b)  is 
added  to  10  parts  of  (a),  and  the  mixture  filtered.  The  mixture  should 
be  made  not  more  than  twenty-four  hours  before  use.  Stain  sections 
for  a  few  minutes  ;  then  decolorise  with  methylated  spirit.  Sometimes 
it  is  advantageous  to  add  to  the  methylated  spirit  a  little  hydrochloric 
acid  (2-3  minims  to  100  c.c. ).  This  staining  solution  is  not  so  much 
used  by  itself,  as  in  Gram's  method,  which  is  presently  to  be  described. 

5.  Carbol-Gcntian-Violet. — 1  part  of  saturated  alcoholic  solution  of 
gentian-violet  is  mixed  with  10  parts  of  5  per  cent  solution  of  carbolic 
acid.     It  is  used  as  No.  4. 

6.  Carbol-Fuchsin  (see  p.  101). — This  is  a  very  powerful  stain,  and, 
when  used  in  the  undiluted  condition,  |-1  minute's  staining  is  usually 
sufficient.     It  is  better,  however,  to  dilute  with  from  five  to  ten  times  its 
volume  of  water  and  stain  for  a  few  minutes.     In  this  form  it  has  a  very 
wide  application.     Methylated  spirit  with  or  without  a  few  drops  of 
acetic  acid  is  the  most  convenient  decolorising  agent.     Then  dehydrate 
thoroughly,  clear,  and  mount. 

Gram's  Method  and  its  Modifications. — In  the  methods 
already  described  the  tissues,  and  more  especially  the  nuclei, 
retain  some  stain  when  decolorisation  has  reached  the  point  to 
which  it  can  safely  go  without  the  bacteria  themselves  being 
affected.  In  the  method  of  Gram,  now  to  be  detailed,  this  does 
not  occur,  for  the  stain  can  here  be  removed  completely  from 
the  ordinary  tissues,  and  left  only  in  the  bacteria.  All  kinds  of 
bacteria,  however,  do  not  retain  the  stain  in  this  method,  and 
therefore  in  the  systematic  description  of  any  species  it  is 
customary  to  state  whether  it  is,  or  is  not,  stained  by  Gram's 
method — by  this  is  meant,  as  will  be  understood  from  what  has 
been  said,  whether  the  particular  organism  retains  the  colour 
after  the  latter  has  been  completely  removed  from  the  tissues.  It 
must,  however,  be  remarked  that  some  tissue  elements  may  retain 
the  stain  as  firmly  as  any  bacteria,  e.g.  keratinised  epithelium, 
calcified  particles,  the  granules  of  mast  cells,  and  sometimes 
altered  red  blood  corpuscles,  etc. 

In  Gram's  method  the  essential  feature  is  the  treating  of  the 
tissue,  after  staining,  with  a  solution  of  iodine.  This  solution  is 
spoken  of  as  Gram's  solution,  and  has  the  following  composition : — 

Iodine         .          .         .         .         .  1  part 

Potassium  iodide          .         .  '       .          2  parts 
Distilled  water  .         .         .      300    „ 

The  following  is  the  method  : — 

1.  Stain  in  aniline  oil  gentian-violet  or  in  carbol-gentian-violet  (vide 
supra)  for  about  five  minutes,  and  wash  in  water. 

2.  Treat  the  section   or  film  with  Gram's   solution  till   its   colour 
becomes  a  purplish  black — generally  about  half  a  minute  or  a  minute  is 
sufficient  for  the  action  to  take  place. 


100  MICROSCOPIC   METHODS 

3.  Decolorise   with  absolute  alcohol   or    methylated   spirit  till    the 
colour  has  almost  entirely  disappeared,  the  tissues  having  only  a  faint 
violet  tint. 

4.  Dehydrate  completely,  clear  with  xylol  and  mount.     In  the  case 
of  film  preparations,  the  specimen  is  simply  washed  in  water,  dried  and 
mounted. 

In  stage  (3)  the  process  of  decolorisation  is  more  satisfactorily 
performed  by  using  clove  oil  after  sufficient  dehydration  with  alcohol, 
the  clove  oil  being  afterwards  removed  by  xylol. 

As  a  contrast  stain  for  the  tissues  carmalum  or  lithia  carmine  is  used 
before  staining  with  gentian-violet  (1).  As  a  contrast  stain  for  other 
bacteria  which  are  decolorised  by  Gram's  method  carbol-fuchsin  diluted 
with  ten  volumes  of  water  or  a  saturated  watery  solution  of  Bismarck  - 
brown  may  be  used  before  stage  (4). 

The  following  modifications  of  Gram's  method  may  be  given : — 

1.  Weigerfs  Modification. — The  contrast  staining  of  the  tissues  and 
stages  (1)  and  (2)  are  performed  as  above. 

(3)  After  using  the  iodine   solution   the    preparation  is  dried   by 
blotting  and  then  decolorised  by  aniline-xylol  (aniline-oil  2,  xylol  1). 

(4)  Wash  well  in  xylol  and  mount  in  xylol  balsam.     Film  preparations 
after  being  washed  in  xylol  may  be  dried  and  thereafter  dilute  carbol- 
fuchsin  may  be  used  to  stain  bacteria  which  have  been  decolorised. 

This  modification  probably  gives  the  most  uniformly  successful  results. 

2.  Nicolles  Modification. — Carbol-gentian-violet  is  used  as  the  stain. 
Treatment   with   iodine  is  carried  out  as  above  and  decolorisation   is 
effected  with  a  mixture  of  acetone  (1  part)  and  alcohol  ^2  parts). 

3.  Kuhnes  Modification. — (1)  Stain  for  five  minutes  in  a  solution 
made  up  of  equal  parts  of  saturated  alcoholic  solution  of  crystal-violet 
("  Krystall- violet ")  and  1  per  cent  solution  of  ammonium  carbonate. 

(2)  Wash  in  water. 

(3)  Place  for  two  to  three  minutes  in  Gram's  iodine  solution,  or  in 
the  following  modification  by  Kiihne  : — 

Iodine 2  parts 

Potassium  iodide        .         .         .         .          4     ,, 
Distilled  water 100     ,, 

For  use,  dilute  with  water  to  make  a  sherry-coloured  solution. 

(4)  Wash  in  water. 

(5)  Decolorise   in   a   saturated   alcoholic    solution  of    fluorescein   (a 
saturated  solution  in  methylated  spirit  does  equally  well). 

(6)  Dehydrate,  clear  and  mount. 

There  is  great  variability  in  the  avidity  with  which  organisms  stained 
by  Gram  retain  the  dye  when  washed  with  alcohol,  and  sometimes 
difficulty  is  experienced  in  saying  whether  an  organism  does  or  does  not 
stain  by  this  method. 

Stain  for  Tubercle  and  other  Acid -fast  Bacilli. — These 
bacilli  cannot  be  well  stained  with  a  simple  watery  solution  of  a 
basic  aniline  dye.  This  fact  can  easily  be  tested  by  attempting 
to  stain  a  film  of  a  tubercle  culture  with  such  a  solution.  They 
require  a  powerful  stain  containing  a  mordant,  and  must  be 
exposed  to  the  stain  for  a  long  time,  or  its  action  may  be  aided 


TUBERCLE   STAINS  101 

by  a  short  application  of  heat.  When  once  stained,  however, 
they  resist  decolorising  even  with  very  powerful  acids ;  they  are 
therefore  called  "acid-fast."  The  smegma  bacillus  also  resists 
decolorising  with  strong  acids  (p.  254),  and  a  number  of  other 
acid-fast  bacilli  have  recently  been  discovered  (p.  252).  Any 
combination  of  gentian-violet  or  fuchsin  with  aniline  oil  or 
carbolic  acid  or  other  mordant  will  stain  the  bacilli  named,  but 
the  following  methods  are  most  commonly  used  : — 

Ziehl-Neehen  Carbol-Fuchsin  Stain. 

Basic  fuchsin  ....  1  part 

Absolute  alcohol      .         .         .         .         10  parts 
Solution  of  carbolic  acid  (1  : 20)        .        100     „ 

1.  Place  the  specimen  in  this  fluid,  and  having  heated  it  till  steam 
rises,  allow  it  to  remain  there  for  five  minutes,  or  allow  it  to  remain  in 
the  cold  stain  for  from  twelve  to  twenty-four  hours.     (Films  and  paraffin 
sections  are  usually  stained  with  hot  stain,  loose  sections  with  cold  ;  in 
hot  stain  the  latter  shrink.) 

2.  Decolorise  with  20  per  cent  solution  of  strong  sulphuric  acid,  nitric 
acid,  or  hydrochloric  acid,  in  water.     In  this  the  tissues  become  yellow. 

3.  Wash  well  with  water.     The  tissues  will  regain  a  faint  pink  tint. 
If  the  colour  is  distinctly  red,  the  decolorisation  is  insufficient,  and  the 
specimen  must  be  returned  to  the  acid.     As  a  matter  of  practice,  it  is 
best  to  remove  the  preparation  from  the  acid  every  few  seconds  and 
wash  in  water,  replacing  the  specimen  in  the  acid  and  re-washing  till 
the  proper  pale  pink  tint  is  obtained.     Then  wash  in  alcohol  for  half  a 
minute  and  replace  in  water. 

4.  Contrast  stain  with  a  saturated  watery  solution  of  rnethylene-blue 
for  half  a  minute,  or  with  saturated  watery  Bismarck-brown  for  from 
two  to  three  minutes. 

5.  Wash  well  with  water.     In  the  case  of  films,  dry  and  mount.     In 
the  case  of  sections,  dehydrate,  clear  and  mount. 

Fraenkel's  Modification  of  the  Ziehl-Neelsen  Stain. 

Here  the  process  is  shortened  by  using  a  mixture  containing 
both  the  decolorising  agent  and  the  contrast  stain. 

The  sections  or  films  are  stained  with  the  carbol-fuchsin  as  above 
described,  and  then  placed  in  the  following  solution  : — 

Distilled  water  .       .        ...         .50  parts 

Absolute  alcohol        .         .         .         .         .     30      ,, 

Nitric  acid 20      ,, 

Methylene-blue  in  crystals  to  saturation. 

They  are  treated  with  this  till  the  red  colour  has  quite  disappeared  and 
been  replaced  by  blue.  The  subsequent  stages  are  the  same  as  in  No. 
5,  supra. 


102  MICROSCOPIC  METHODS 

Leprosy  bacilli  are  stained  in  the  same  way,  but  are  rather 
more  easily  decolorised  than  tubercle  bacilli,  and  it  is  better  to 
use  only  5  per  cent  sulphuric  acid  in  decolorising. 

In  the  case  of  specimens  stained  either  by  the  original  Ziehl- 
Neelsen  method,  or  by  Fraenkel's  modification,  the  tubercle  or 
leprosy  bacilli  ought  to  be  bright  red,  and  the  tissue  blue  or 
brown,  according  to  the  contrast  stain  used.  Other  bacteria 
which  may  be  present  are  also  coloured  with  the  contrast  stain. 

The  Staining  of  Spores. — If  bacilli  containing  spores  are 
stained  with  a  watery  solution  of  a  basic  aniline  dye  the  spores 
remain  unstained.  The  spores  either  take  up  the  stain  less 
readily  than  the  protoplasm  of  the  bacilli  or  they  have  a  resisting 
envelope  which  prevents  the  stain  penetrating  to  the  protoplasm. 
Like  the  tubercle  bacilli,  when  once  stained  they  retain  the  colour 
with  considerable  tenacity.  The  following  is  the  simplest  method 
for  staining  spores  > — 

1.  Stain  cover-glass  films  as  for  tubercle  bacilli. 

2.  Decolorise  with  1  per  cent  sulphuric  acid  in  water  or  with  methyl- 
ated spirit.     This  removes  the  stain  from  the  bacilli. 

3.  Wash  in  water. 

4.  Stain  with  saturated  watery  methylene-blue  for  half  a  minute. 

5.  Wash  in  water,  dry,  and  mount  in  balsam. 

The  result  is  that  the  spores  are  stained  red,  the  protoplasm  of  the 
bacilli  blue. 

The  spores  of  some  organisms  lose  the  stain  more  readily  than  those 
of  others,  and  for  some,  methylated  spirit  is  a  sufficiently  strong 
decolorising  agent  for  use.  If  sulphuric  acid  stronger  than  1  per  cent 
is  used  the  spores  of  many  bacilli  are  readily  decolorised. 

Hollers  Method. — The  following  method,  recommended  by  Holier,  is 
much  more  satisfactory  than  the  previous.  Before  being  stained,  the  films 
are  placed  in  chloroform  for  2  minutes,  and  then  in  a  5  per  cent  solution 
of  chromic  acid  for  ^-2  minutes,  the  preparation  being  well  washed 
after  each  reagent.  Thereafter  they  are  stained  and  decolorised  as  above. 

The  Staining  of  Capsules. — The  two  following  methods  may 
be  recommended  in  the  case  of  capsulated  bacteria  : — 

(a)  Welch's  Method. — This  depends  on  the  fact  that  in  many  cases 
the  capsules  can  be  fixed  with  glacial  acetic  acid. 

Films  when  still  wet  are  placed  in  this  acid  for  a  few  seconds. 

The  superfluous  acid  is  removed  with  filter-paper  and  the  preparation 
is  treated  with  gentian-violet  in  aniline  oil  water  repeatedly  till  all  the 
acetic  acid  is  removed. 

Then  wash  with  1-2  per  cent  solution  of  sodium  chloride  and  examine 
in  the  same  solution. 

The  capsule  appears  as  a  pale  violet  halo  around  the  deeply  stained 
bacterium. 

(6)  Richard  Muirs  Method  (as  recently  modified). 

1.  The  film  containing  the  bacteria  must  be  very  thin.  It  is  dried 
and  stained  in  filtered  carbol-iuchsin  for  half  a  minute,  the  preparation 
being  gently  heated. 


STAINING   OF   FLAGELLA  103 

2.  Wash  slightly  with  spirit  and  then  well  in  water. 

3.  Place  in  following  mordant  for  a  few  seconds  :  — 

Saturated  solution  of  corrosive  sublimate     .         .         .2  parts 
Tannic  acid  solution  —  20  per  cent        .         .         .         .     2     ,, 
Saturated  solution  of  potash  alum        ...         .         .     5     ,, 

4.  Wash'  well  in  water. 

5.  Treat  with  methylated  spirit  for  about  a  minute. 
The  preparation  has  a  pale  reddish  appearance. 

6.  Wash  well  in  water. 

7.  Counterstain  with  watery  solution  of  ordinary  methylene-blue  for 
half  a  minute. 

8.  Dehydrate  in  alcohol,  clear  in  xylol,  and  mount  in  balsam. 

The  bacteria  are  a  deep  crimson  and  the  capsules  of  a  blue  tint.  The 
capsules  of  bacteria  in  cultures  may  sometimes  be  demonstrated  by  this 
method. 

The  Staining  of  Flagella.  —  The  staining  of  the  flagella  of 
bacteria  is  the  most  difficult  of  all  bacteriological  procedures, 
and  it  requires  considerable  practice  to  ensure  that  good  results 
shall  be  obtained.  Many  methods  have  been  introduced,  of 
which  the  two  following  are  the  most  satisfactory. 

Preparation  of  Films.  —  In  all  the  methods  of  staining 
flagella,  young  cultures  on  agar  should  be  used,  say  a  culture 
incubated  for  from  ten  to  eighteen  hours  at  37°  C.  A  very 
small  portion  of  the  growth  is  taken  on  the  point  of  a  platinum 
needle  and  carefully  mixed  in  a  little  water  in  a  watch-glass  ; 
the  amount  should  be  such  as  to  produce  scarcely  any  turbidity 
in  the  water.  A  film  is  then  made  by  placing  a  drop  on  a 
clean  cover-glass  and  carefully  spreading  it  out  with  the  needle. 
It  is  allowed  to  dry  in  the  air  and  is  then  passed  twice  or  thrice 
through  a  flame,  care  being  taken  not  to  over-heat  it.  The 
cover-glasses  used  should  always  be  cleaned  in  the  mixture  of 
sulphuric  acid  and  potassium  bichromate  described  on  page  87. 

1.  Pitfield's  Method  as  modified  by  Richard  Muir. 
Prepare  the  following  solutions  :  — 
A.    The  Mordant. 

Tannic  acid,  10  per  cent  watery  solution,  filtered    .     10  c.c. 


Corrosive  sublimate,  saturated  watery  solution 


Alum,  saturated  watery  solution     .         .         ;  5    ,, 

Carbol-fuchsin  (vide  p.  101)     .....       5    ,, 

Mix  thoroughly.  A  precipitate  forms,  which  must  be  allowed  to 
deposit,  either  by  centrifugalising  or  simply  by  allowing  to  stand. 
Remove  the  clear  fluid  with  a  pipette  and  transfer  to  a  clean  bottle. 
The  mordant  keeps  well  for  one  or  two  weeks. 

B.   The  Stain. 

Alum,  saturated  watery  solution     .         .         .         .     10  c.c. 
Gentian-  violet,  saturated  alcoholic  solution     .         .       2    ,, 


104  MICROSCOPIC   METHODS 

The  stain  should  not  be  more  than  two  or  three  days  old  when  used. 
It  may  be  substituted  in  the  mordant  in  place  of  the  carbol-fuchsin. 

The  film  having  been  prepared  as  above  described,  pour  over  it  as 
much  of  the  mordant  as  the  cover-glass  will  hold.  Heat  gently  over  a 
flame  till  steam  begins  to  rise,  allow  to  steam  for  about  a  minute,  and 
then  wash  well  in  a  stream  of  running  water  for  about  two  minutes. 
Then  dry  carefully  over  the  flame,  and  when  thoroughly  dry  pour  on 
some  of  the  stain.  Heat  as  before,  allowing  to  steam  for  about  a  minute, 
wash  well  in  water,  dry  and  mount  in  a  drop  of  xylol  balsam. 

This  method  has  yielded  the  best  results  in  our  hands. 


2.    Tan  Ermengenis  Method  for  Staining  Flagella. 

The  films  are  prepared  as  above  described.  Three  solutions  are  here 
necessary : — 

Solution  A.     (Bain  fixateur] — 

Osmic  acid,  2  per  cent  solution          .         .  .         1  part 

Tannin,  10-25  per  cent  solution          ....         2  parts 

Place  the  films  in  this  for  one  hour  at  room  temperature,  or  heat  over 
a  flame  till  steam  rises  and  keep  in  the  hot  stain  for  five  minutes. 
Wash  with  distilled  water,  then  with  absolute  alcohol  for  three  to  four 
minutes,  and  again  in  distilled  water,  and  treat  with 

Solution  B.     (Bain  sensibilisateur] — 

*5  per  cent  solution  of  nitrate  of  silver  in  distilled  water.  Allow 
films  to  be  in  this  a  few  seconds.  Then  without  washing  transfer  to 

Solution  C.     (Bain  reducteur  et  reinfor$ateur] — 

Gallic  acid         .         .         .  .  .  .  .  w>   «  5  grm. 

Tannin      .         .         .         .  .  .  .  .  .  3     ,, 

Fused  potassium  acetate  .  .  .  .  .  .  10     ,, 

Distilled  water          .         .  .  .  .  .  350  c.c. 

Keep  in  this  for  a  few  seconds.  Then  treat  again  with  Solution  B  till 
the  preparation  begins  to  turn  black.  Wash,  dry,  and  mount. 

It  is  better,  as  Mervyn  Gordon  recommends,  to  leave  the  specimen  in 
B  for  two  minutes  and  then  to  transfer  to  C  for  one  and  a  half  to  two 
minutes,  and  not  to  transfer  again  to  B.  It  will  also  be  found  an 
advantage  to  use  a  fresh  supply  of  C  for  each  preparation,  a  small 
quantity  being  sufficient.  The  beginner  will  find  the  typhoid  bacillus 
or  the  bacillus  coli  communis  very  suitable  organisms  to  stain  by  this 
method. 

Although  the  results  obtained  by  this  method  are  sometimes  excellent, 
they  vary  considerably.  Frequently  both  the  organisms  and  flagella 
appear  of  abnormal  thickness.  This  is  due  to  the  fact  that  the  process 
on  which  the  method  depends  is  a  precipitation  rather  than  a  true 
staining.  The  pictures  on  the  whole  are  less  faithful  than  in  the  first 
method. 

Staining  of  Spirochsete  in  Sections. — The  following  im- 
pregnation method,  which  is  practically  that  of  Ramon-y-Cajal 


THE   ROMANOWSKY   STAINS  105 

for  nerve  fibrillae,  has  been  applied  for  this  purpose  by  Levaditi 
and  gives  excellent  results. 

(1)  The  tissues,  which  ought  to  be  in  thin  slices,  about  1  mm. 
in  thickness,  are  best  fixed  in  10  per  cent  formalin  solution  for 
twenty-four  hours. 

(2)  They  are  washed  for  an  hour  in  water  and  then  brought 
into  96  per  cent  alcohol  for  twenty-four  hours. 

(3)  They  are  then  placed  in  1*5  per  cent  solution  of  nitrate 
of  silver  in  a  dark  bottle,  and  are  kept  in  an  incubator  at  37°  C. 
for  three  days. 

(4)  They  are  washed  in  water  for  about  twenty  minutes,  and 
are  thereafter  placed  in  the  following  mixture,  namely  : — 

Pyrogallic  acid,  4  parts. 

Formalin,  5  parts. 

Distilled  water  up  to  100  parts. 

They  are  kept  in  this  mixture  in  a  dark  bottle  for  forty-eight 
hours  at  room  temperature. 

(5)  They  are  then  washed  in  water  for  a  few  minutes,  taken 
through    increasing    strengths    of    alcohol,   and    embedded    in 
paraffin  in  the  usual  way.     The  sections  ought  to  be  as  thin  as 
possible.     In  satisfactory  preparations  the  spirochaetes  appear  of 
an  almost  black  colour  against  the  pale  yellow  background  of 
the  tissues.     The  latter  can  be  contrast  stained  by  weak  carbol- 
fuchsin  or  by  toluidin  blue. 

(For  the  staining  of  spirochaetes  in  films  see  p.  107.) 

The  Romanowsky  Stains. — Within  recent  years  the  numerous 
modifications  of  the  Eomanowsky  stain  have  been  extensively 
used.  The  dye  concerned  is  the  compound  which  is  formed 
when  watery  solutions  of  medicinal  methylene-blue  and  water- 
soluble  eosin  are  brought  together.  This  compound  is  insoluble 
in  water  but  soluble  in  alcohol — the  alcohol  employed  being 
methyl  alcohol.  The  stain  was  originally  used  by  Romanowsky 
for  the  malarial  parasite,  and  its  special  quality  is  that  it 
imparts  to  certain  elements,  such  as  the  chromatin  of  this 
organism,  a  reddish-purple  hue.  This  was  at  first  thought  to  be 
simply  due  to  the  combination  of  the  methylene-blue  and  the 
eosin,  but  it  is  now  recognised  that  certain  changes,  such  as 
occur  in  methylene-blue  solutions  with  age,  are  necessary.  In 
the  modern  formulae  these  changes  are  brought  about  by 
treatment  with  alkalies,  especially  alkaline  carbonates,  as  was 
first  practised  by  Unna  in  the  preparation  of  his  polychrome 
methylene-blue.  It  is  not  certainly  known  to  what  particular 


106  MICROSCOPIC   METHODS 

new  body  the  reddish  hue  is  due,  but  it  may  be  to  methyl-violet 
or  to  methyl-azure,  both  of  which  result  from  the  action  of  alkali 
on  methylene-blue.  The  stains  are  much  used  in  staining  blood- 
films  (in  which  the  characters  of  both  nucleus  and  cytoplasm  are 
beautifully  brought  out),  in  staining  bacteria  in  tissues  or 
exudates,  the  malaria  parasite,  trypanosomes,  the  pathogenic 
spirochsetes  (such  as  the  spirochaete  pallida),  and  protozoa 
generally. 

The  following  are  the  chief  formulae  in  use  : — 

1.  Jenner's  Stain. — This  is  an  excellent  blood  stain,  but  is  not  so  good 
for   the   study   of  parasites   as   the   others   to   be   mentioned.     In   its 
preparation  no  alkali  is  used.     It  is  made  by  mixing  equal  parts  of  (a) 
a  1  '2  to  1  '25  per  cent  solution  of  Griibler's  water  soluble  eosin  (yellow 
shade)  in  distilled  water  and  (b)  1  per  cent  Griibler's  medicinal  methy- 
lene-blue (also  a  watery  solution).      The  mixture  is  allowed  to  stand 
twenty-four  hours,  is  filtered,  and  the  residue  is  dried  at  55°  C.  ;  the 
powder  is  shaken  up  in  distilled  water,  filtered,  washed  with  distilled 
water  and  dried.    Of  the  powder,  5  grms.  are  dissolved  in  100  c.c.  Merck's 
methyl  alcohol.     For  use  a  few  drops  are  placed  on  the  dried  unfixed 
film  for  one  to  three  minutes,  the  dye  is  poured  off,  and  the  preparation 
washed  with  distilled  water  till  it  presents  a  pink  colour  ;   it  is  then 
dried  between  filter-paper  and  mounted  in  xylol  balsam. 

2.  Leishmaris  Stain. — The  following  solutions  are  prepared  :  (a)  to 
a  1  per  cent  solution  of  medicinal  methylene-blue  is  added  '5  per  cent 
sodium  carbonate  ;  the  mixture  is  kept  at  65°  C.  for  twelve  hours  and 
then  for  ten  days  at  room  temperature  ('25  per  cent  formalin  may  be 
added  as  a  preservative)  ;   (b)  1-1000  solution  of  eosin,   extra  B.A.,  in 
distilled  water.     Equal  volumes   of  the  two  solutions  are  mixed  and 
allowed  to  stand  for  six  to  twelve  hours  with  occasional  stirring,   the 
precipitate  is  collected,  filtered,  washed  with  distilled  water  and  dried. 
For  use   '15    per  cent    is  dissolved  in  Merck's  methyl  alcohol  ("for 
analysis,  acetone  free ")  as  follows  :   the  powder  is   placed  in  a  clean 
mortar,  a  little  of  the  alcohol  is  added  and  well  rubbed  up  with    a 
pestle ;   the  undissolved   powder   is    allowed   to   settle  and   the   fluid 
decanted  into  a  dry  bottle  ;  the  process  is  repeated  with  fresh  fractions 
of  the  solvent  till  practically  all  the  stain  is  dissolved,  and  the  bottle 
is  well  stoppered  ;    the   stain  will   keep  for  a   long   period.     For  the 
staining  of  films  a  few  drops  of  the  stain  are  placed  on  the  unfixed 
preparation    for   fifteen   to   thirty  seconds   so    as    to   cover    it   with    a 
shallow  layer   (the   stain   may  be   conveniently  spread    over    the  film 
with  a  glass   rod)  and  the  film  is  tilted  to  and  fro  so  as  to  prevent 
drying.     This  treatment  efficiently  fixes  the  film  by  the  action  of  the 
methyl  alcohol.     About  double  the  quantity  of  distilled  water  is  now 
dropped  on  the  film,  and  the  stain  and  diluent  are  quickly  mixed  with 
the  rod.     Five  minutes  are  now  allowed  for  staining,  and  the  stain  is 
then  gently  washed  off  with  distilled  water.     A  little  of  the  water  is 
kept  on  the  film  for  half  a  minute  to  intensify  the  colour  contrasts  in 
the  various  cells.     For  certain  special  structures  such  as  SchiifFner's  dots 
or  Maurer's  dots  in  the  malarial  parasite  a  longer  staining  (up  to  one 
hour)  may  be  necessary,  and  in  any  case  it  is  well  to  practise  being  able 
to  control  the  depth  of  the  staining  effect  by  observation  with  a  low 
power  objective.     If  a  preparation  is  to  be  stained  for  a  long  time  it 


THE   KOMANOWSKY   STAINS  107 

must  be  kept  covered,  and  if  in  such  cases  a  granular  deposit  is  formed 
this  may  be  got  rid  of  by  a  quick  wash  with  absolute  alcohol.  If  in  blood 
films  the  red  corpuscles  appear  bluish  instead  of  pink  the  colour  may 
be  restored  by  washing  the  film  with  acetic  acid,  1-1500.  The  film  is 
dried  between  niter-paper  and  mounted. 

For  staining  sections  a  little  modification  is  necessary.  A  paraffin 
section  is  taken  into  distilled  water  as  usual,  the  excess  of  water  is  drained 
off,  and  a  mixture  of  one  part  of  stain  and  two  parts  of  distilled  water 
is  placed  on  it.  The  stain  is  allowed  to  act  for  five  to  ten  minutes  till  the 
tissue  appears  a  deep  Oxford  blue  ;  it  is  then  decolorised  with  1-1500 
acetic  acid — the  effect  being  watched  under  a  low-power  lens.  The  blue 
begins  to  come  out,  and  the  process  is  allowed  to  go  on  till  only  the 
nuclei  remain  blue.  The  section  is  then  washed  with  distilled  water, 
rapidly  dehydrated  with  alcohol,  cleared  and  mounted.  If,  as  some- 
times happens,  the  eosin  tint  be  too  well  marked  it  can  be  lightened 
by  the  action  of  1-7000  solution  of  caustic  soda,  this  being  washed  off 
whenever  the  desired  colour  has  been  attained. 

3.  J.  H.  Wright's  Stain. — In  this  modification  1  per  cent  methylene- 
blue  (BX  or  Ehrlich's  rectified)  and  \  per  cent  sodium  carbonate  (both 
in  water)  are  mixed  and  placed  in  a  Koch's  steriliser  for  an  hour.     When 
the  fluid  is  cold,  1-1000  solution  of  extra  B.A.  eosin  is  added  till  the  mixture 
becomes  purplish  and  a  finely  granular   black  precipitate  appears   in 
suspension  (about  500  c.c.  eosin  to  100  c.c.  methylene-blue  solution  are 
required)  ;  the  precipitate  is  filtered  off  and  dried  without  being  washed. 
A  saturated  solution  of  this  is  made  in  the  pure  methyl  alcohol ;  this  is 
filtered  and  diluted  by  adding  to  80  c.c.  of  the  saturated  solution  20  c.c. 
of  methyl  alcohol.     The  application  of  the  stain  is  almost  the  same  as 
with  Leishman's.     A  few  drops  are  placed  on  the   preparation  for  a 
minute  for  fixation  ;    water  is  then  dropped  on  till  a  green  iridescent 
scum  appears  on  the  top  of  the  fluid  and  staining  goes  on  for  about 
two  minutes  ;  the  stain  is  then  washed  off  with  distilled  water  and  a 
little  is  allowed  to  remain  on  the  film  till  differentiation  is  complete  ; 
the  preparation  is  carefully  dried  with  filter-paper  and  mounted. 

4.  Giemsa' s  Stain.  —  Giemsa   believes   that    the    reddish-blue   hue 
characteristic  of  the   Romanowsky  stain   is   due   to  the    formation  of 
methyl-azure,  and  he  has  prepared  this  by  a  method  of  his  own  under 
the   name  "  Azur  I."     From  this,   by    the  addition    of  equal  parts  of 
medicinal  methylene-blue,  he  prepares  what  he  calls  "Azur  II.,"  and 
from  this  again  by  the  addition  of  eosin  he  prepares  "  Azur  II. -eosin." 
The  latest  formula  for  the  finished  stain  is  as  follows  : — Azur  II. -eosin  3 
gr.,  Azur  II.  8  gr.,  Glycerin  (Merck,  chemically  pure)  250  gr.,  Methyl 
alcohol  (Kahlbaum,  I.)  250  gr.     This  stain  has  been  extensively  used 
for  demonstrating  the  Spirochsete  pallida,  but  it  can  be  used  for  any 
other  purpose  to  which  the  Romanowsky  stains  are  applicable.     For  the 
spirochsete  the  following  are  Giemsa's  directions  :  — 

(1)  Fix  films  in  absolute  alcohol  for  fifteen  to  twenty  minutes,  dry 
with  filter-paper.  (2)  Dilute  stain  with  distilled  water — one  drop  of 
stain  to  1  c.c.  water  (the  mixture  being  well  shaken).  (Sometimes  the 
water  is  made  alkaline  by  the  addition  of  one  drop  of  1  per  cent  potassium 
carbonate  to  10  c.c.  water).  (3)  Stain  for  fifteen  minutes.  (4)  Wash  in 
brisk  stream  of  distilled  water.  (5)  Drain  with  filter-paper,  dry,  and 
mount  in  Canada  balsam. 

With  regard  to  the  Jenner  and  Giemsa  stains  it  is  best  to  obtain  the 
solutions  from  Griibler  ready  for  use  ;  the  powder  for  Leishman's  stain 
may  be  obtained  from  the  same  source  and  the  solution  made  up  by 


108 


MICROSCOPIC   METHODS 


the  worker  himself.  Cabot  states  that  Wright's  stain  can  be  obtained 
from  the  Harvard  Co-operative  Society,  Boylston  Street,  Boston,  U.S.A. 
Neisser's  Stain.  —  Neisser  introduced  the  following  stain  as  an  aid  to 
the  diagnosis  of  the  diphtheria  bacillus.  Two  solutions  are  used  as 
follows  :  (a)  1  grm.  methylene-blue  (Griibler)  is  dissolved  in  20  c.c.  of 
96  per  cent  alcohol,  and  to  the  solution  are  added  950  c.c.  of  distilled 
water  and  50  c.c.  of  glacial  acetic  acid  ;  (&)  2  grins.  Bismarck-brown 
(vesuvin)  dissolved  in  a  litre  of  distilled  water.  Films  are  stained  in 
(a)  for  1-3  seconds  or  a  little  longer,  washed  in  water,  stained  for  3-5 
seconds  in  (&),  dried,  and  mounted.  The  protoplasm  of  the  diphtheria 
bacillus  is  stained  a  faint  brown  colour,  the  granules  a  blue  colour. 
Neisser  considers  that  this  reaction  is  characteristic  of  the  organism, 
provided  that  cultures  on  Loffler's  serum  are  used  and  examined  after 
9-24  hours'  incubation  at  34-35°  C.  Satisfactory  results  are  not  always 
obtained  in  the  case  of  films  prepared  from 
membrane,  etc.  ,  but  there  is  no  doubt  that  here 
also  the  method  is  one  of  considerable  value. 

SPECIAL  BACTEKIOLOGICAL  METHODS. 

Wright's  Methods  of  measuring  small 
amounts  of  Fluids.  —  In  ordinary  work  fine 
calibrated  pipettes  may  be  used  for  measur- 
ing small  quantities  of  fluids,  but  such 
pipettes  are  not  always  available,  and  by 
Wright's  technique  if  a  Gower's  5  c.mm. 
haemocytometer  pipette  be  at  hand  any 
measurements  may  be  undertaken,  —  in  fact, 
once  the  pipette  now  to  be  described  (see 
Fig.  45)  is  made  we  are  independent  of 
other  means  of  measurement.  A  piece  of 
quill  tubing  is  drawn  out  to  capillary 
dimensions,  and  the  extreme  tip  of  it  is 
heated  in  a  peep  flame  and  then  drawn  out 
till  it  is  of  the  thickness  of  a  hair  though 
still  possessing  a  bore.  If  the  point  be 
c  mm  pipette  A  broken  off  this  hair  and  mercury  be  run 
Casing  of  quill  tubing';  into  the  tube  the  metal  will  be  caught 
B,  rubber  nipple  ;  C,  where  the  tube  narrows  and  will  pass  no 
wax  luting  ;  E  to  F  f  urther  —  in  fact,  though  air  will  pass, 


---B 

--D 
F 

—  -A 

FIG.    4 

\l 

C 

c- 

5.  —  Wright's    5 

E,  hair  capillary.          this  tube  5   c.mm.  of   mercury,  measured 
from  a  Gower's  pipette,  is  run  down  till 

it  will  go  no  further.  A  mark  is  made  on  the  tube  at  the 
proximal  end  of  the  mercury,  which  is  now  allowed  to  run  out, 
and  the  tube  is  carefully  cut  through  at  the  mark.  A  piece 
of  ordinary  quill  tubing  is  drawn  out  and  broken  off  just 
below  where  its  narrowing  has  begun,  the  capillary  tube  has 


TESTING   OF   PROPERTIES   OF   SERUM        109 

some  wax  moulded  round  its  middle,  the  hair  end  is  slipped 
through  the  broken-off  end  just  mentioned,  and  the  tube  is  fixed 
in  position  as  shown  in  the  figure.  A  rubber  nipple  placed  on 
the  end  of  the  pipette  completes  the  apparatus.  If  by  pressing 
the  nipple  the  air  be  expelled  from  the  pipette  and  the  end 
dipped  under  mercury,  exactly  5  c.mm.  will  be  taken  up.  Thus 
when  pressure  on  the  nipple  is  relaxed,  other  tubes  can  be  very 
readily  calibrated  by  the  mercury  being  expelled  into  them  and 
its  limits  marked  on  their  bores. 

For  measuring  equal  parts  of  different  fluids  the  pipette 
described  in  connection  with  agglutination  is  very  useful 
(see  Fig.  46  d). 


The  Testing  of  Agglutinative  and  Sedimenting  Properties 
of  Serum. 

By  agglutination  is  meant  the  aggregation  into  clumps  of 
uniformly  disposed  bacteria  in  a  fluid ;  by  sedimentation  the 
formation  of  a  deposit  composed  of  such  clumps  when  the  fluid 
is  allowed  to  stand.  Sedimentation  is  thus  the  naked-eye  evidence 
of  agglutination.  The  blood  serum  may  acquire  this  clumping 
power  towards  a  particular  organism  under  certain  conditions ; 
these  being  chiefly  met  with  when  the  individual  is  suffering 
from  the  disease  produced  by  the  organism,  or  has  recovered  from 
it,  or  when  a  certain  degree  of  immunity  has  been  produced 
artificially  by  injections  of  the  organism.  The  nature  of  this 
property  will  be  discussed  later.  Here  we  shall  only  give  the 
technique  by  which  the  presence  or  absence  of  the  property  may 
be  tested.  There  are  two  chief  methods,  a  microscopic  and  a 
naked  eye,  corresponding  to  the  effects  mentioned  above.  In 
both,  the  essential  process  is  the  bringing  of  the  diluted  serum 
into  contact  with  the  bacteria  uniformly  disposed  in  a  fluid.  In 
the  former  this  is  done  on  a  glass  slide,  and  the  result  is  watched 
under  the  microscope ;  the  occurrence  of  the  phenomenon  is 
shown  by  the  aggregation  of  the  bacteria  into  clumps,  and  if  the 
organism  is  motile  this  change  is  preceded  or  accompanied  by 
more  or  less  complete  loss  of  motility.  In  the  latter  method 
the  mixture  is  placed  in  an  upright  thin  glass  tube ;  sediment- 
ation is  shown  by  the  formation  within  a  given  time  (say  12  or 
24  hours)  of  a  somewhat  flocculent  layer  at  the  bottom,  the  fluid 
above  being  clear.  Two  points  should  be  attended  to :  (a) 
controls  should  always  be  made  with  normal  serum,  and  (6)  the 
serum  to  be  tested  should  never  be  brought  in  the  undiluted 


110 


MICROSCOPIC   METHODS 


condition  into  contact  with    the  bacteria.     The  stages  of  pro- 
cedure are  the  following  : — 

1.   Blood  is  conveniently  obtained  by  pricking  the  lobe  of  the  ear, 
which  ..should  previously  have  been  washed  with  a  mixture  of  alcohol 

and  ether  and  allowed  to 
dry.  The  blood  is  drawn 
up  into  a  Wright's  blood 
capsule  (Fig.  47)  or  into 
the  bulbous  portion  of  a 
capillary  pipette,  such  as 
in  Fig.  46,  a.  (These  pip- 
ettes can  be  readily  made 
by  drawing  out  quill  glass 
tubing  in  a  flame.  It  is 
convenient  always  to  have 
several  ready  for  use.) 
The  pipette  is  kept  in  the 
upright  position,  one  end 
being  closed.  For  purposes 
of  transit,  break  off  the 
bulb  at  the  constriction 
and  seal  the  ends.  After 
the  serum  has  separated 
from  the  coagulum  the 
bulb  is  broken  through 
near  its  upper  end  and  the 
serum  removed  by  means 
of  another  capillary  pip- 
ette. The  serum  is  then 
to  be  diluted. 

2.  The  serum  may  be 
diluted  (a)  by  means  of  a 
graduated  pipette — either 
a  leucocytometer  pipette 
(Fig.  46,  b]  or  some  corre- 
sponding form.  In  this 
way  successive  dilutions 
of  1  :  10,  1  :  20,  1  :  100, 
etc.,  can  be  rapidly  made. 
This  is  the  best  method. 

,  (&)  By  means  of  a  capillary 

\  /  -i-  n         pipette  with  a  mark  on  the 

tube,  the  serum  is  drawn 
d 


FIG.  46. — Tubes  used  in  testing  agglutinating  and 
sedimenting  properties  of  serum. 


up  to  the  mark  and  then 
blown  out  into  a  glass 
capsule  ;  equal  quantities 
of  bouillon  are  successively 
measured  in  the  same  way 
and  added  till  the  requisite  dilution  is  obtained,  (c)  By  means  of  a 
platinum  needle  with  a  loop  at  the  end  (Delepine's  method).  A  loopful 
of  serum  is  placed  on  a  slide  and  the  desired  number  of  similar  loopfuls 
of  bouillon  are  separately  placed  around  on  the  slide.  The  drops  are 
then  mixed. 

A  very  convenient  and  rapid  method  of  combining  the  steps  1  and  2 


THE   OPSONIC   TECHNIQUE  111 

is  to  draw  a  drop  of  blood  up  to  the  mark  1  or  '5  on  a  leucocytometer 
pipette  and  draw  the  bouillon  after  it  till  the  bulb  is  filled.  A  dilution 
of  10  or  20  times  is  thus  obtained.  Then  blow  the  mixture  into  a 
U-shaped  tube  (Fig.  46,  c)  and  centrifugalise  or  simply  allow  the  red 
corpuscles  to  separate  by  standing.  (In  this  method  of  course  the 
dilution  is  really  greater  than  if  pure  serum  were  used,  and  allowance 
must  therefore  be  made  in  comparing  results.)  The  presence  of  red 
corpuscles  is  no  drawback  in  the  case  of  the  microscopic  method,  but  when 
sedimentation  tubes  are  used  the  corpuscles  should  be  separated  first. 

3.  The  bacteria  to  be  tested  should  be  taken  from  young  cultures, 
preferably  not  more  than  twenty-four  hours  old,  incubated   at  37°  C. 
They  may  be  used  either  as  a  bouillon  culture  or  as  an  emulsion  made 
by  adding  a  small  portion  of  an  agar  culture  to  bouillon.     In  the  latter 
case  the  mass  of  bacteria  on  a  platinum  loop  should  be  gently  broken 
down  at   the   margin   of  the  fluid  in   a   watch-glass.     When   a   thick 
turbidity  is   thus   obtained,   any  remaining  fragments  should   first   be 
removed  and  then  the  organisms  should  be  uniformly  mixed  with  the 
rest  of  the   fluid.     The  bacterial   emulsion  ought  to  have  a  faint  but 
distinct  turbidity.     (When  the  exact  degree  of  sedimenting  power  of  a 
serum  is  to  be  tested — expressed  as  the  highest  dilution  in  which   it 
produces  complete  sedimentation  within  twenty-four  hours — a  standard 
quantity  (by  weight)  of  bacteria  must  be  added  to  a  given  quantity  of 
bouillon.     This  is  not  necessary  for  clinical  diagnosis.) 

4.  To  test  microscopically,    mix   equal  quantities   (measured    by   a 
marked    capillary   pipette)    of    the    diluted    serum    and    the    bacterial 
emulsion  on  a  glass  slide,  cover  with  a  cover-glass,  and  examine  under 
the  microscope.     The  form  of  glass   slide  used  for  hang-drop  cultures 
(Fig.  27)  will  be  found   very  suitable.     The  ultimate   dilution  of  the 
serum  will,  of  course,  be  double  the  original  dilution. 

To  observe  sedimentation  mix  equal  parts  of  diluted  serum  and  of 
bacterial  emulsion  and  place  in  a  thin  glass  tube — a  simple  tube  with 
closed  end  or  a  U-tube.  Keep  in  upright  position  for  twenty-four 
hours.  One  of  Wright's  sedimentation  tubes  is  shown  in  Fig.  46,  d. 
Diluted  serum  is  drawn  up  to  fill  the  space  mn,  a  small  quantity  of  air 
is  sucked  up  after  it  to  separate  it  from  the  bacterial  emulsion,  which 
is  then  drawn  up  in  the  same  quantity  ;  the  diluted  serum  will  then 
occupy  the  position  Tel.  The  fluids  are  then  drawn  several  times  up 
into  the  bulb  and  returned  to  the  capillary  tube  so  as  to  mix,  and  finally 
blown  carefully  down  close  to  the  lower  end,  which  is  then  sealed  oft'. 
The  sediment  collects  at  the  lower  extremity. 

It  may  be  said  that  it  is  often  important  to  observe  not  only  the 
strongest  concentration  of  a  serum  which  will  produce  agglutination  but 
also  the  weakest. 

Method  of  measuring  the  Phagocytic  Capacity  of  the 
Leucocytes — the  Opsonic  Technique. — This  was  first  done 
by  Leishman  by  a  very  simple  method  as  follows  :  A  piece 
of  quill  tubing  is  drawn  out  to  a  capillary  diameter  so  as 
to  make  a  pipette  about  six  inches  long.  The  point  is 
broken  off  and  a  rubber  nipple  adjusted  to  the  wide  end,  a 
mark  is  made  with  an  oil  pencil  about  three-quarters  of  an 
inch  above  the  orifice.  Blood  is  drawn  from  the  finger  up 
to  the  mark,  then  an  air-bubble  is  allowed  to  pass  in.  A  thin 


112  MICROSCOPIC   METHODS 

emulsion  of  the  bacterium  to  be  tested  having  been  prepared,  a 
quantity  of  this  is  also  drawn  up  to  the  mark.  The  two  fluids 
are  then  thoroughly  mixed  by  being  first  blown  out  on  to  a 
sterile  slide  and  then  being  drawn  back  into  the  pipette  and 
expelled, — this  being  repeated  several  times.  A  cover-glass  is 
placed  over  the  drop,  and  the  slide  is  placed  in  the  incubator  at 
37°  C.  for  fifteen  minutes.  The  cover-glass  is  then  slipped  off 
so  as  to  make  a  film  preparation  which  in  the  case  of  ordinary 
bacteria  may  be  stained  by  Irishman's  method.  The  number 
of  bacteria  present  in,  say,  50  polymorphonuclear  cells  succes- 
sively examined  is  determined  and  an  average  struck.  The 
method  was  first  used  for  showing  that  in  cases  of  staphylococcus 
infection  the  average  number  of  bacteria  taken  up  was  less  than 
in  a  control  in  which  the  same  bacterial  emulsion  was  exposed 
to  the  blood  of  a  healthy  individual.  In  making  such  an 
observation  drops  from  the  two  mixtures  are  placed  on  the  same 
slide  under  separate  cover-glasses  and  the  preparation  incubated. 
One  cover  is  then  slipped  to  one  end  of  the  slide  and  the  other  to 
the  other, — the  two  films  being  then  stained  as  one. 

According  to  Wright's  view  the  process  of  phagocytosis  in 
blood  outside  the  body  is  not  a  simple  one,  and^,  before  a 
leucocyte  takes  up  a  bacterium  the  latter  must  be  acted  on 
in  some  way  by  substances  present  in  the  serum,  which  Wright 
calls  opsonins  (see  Immunity).  The  technique  by  which  the 
actions  of  these  opsonins  is  studied  has  been  elaborated  by 
Wright  and  his  co-workers  in  connection  with  his  work  on 
bacterial  vaccines,  especially  in  relation  to  infection  by  the 
pyogenic  cocci  and  the  tubercle  bacillus.  This  technique 
involves  (1)  the  preparation  of  the  bacterial  emulsion,  (2)  the 
preparation  of  the  leucocytes,  (3)  the  preparation  of  samples  of 
(a)  serum  from  a  normal  person,  (6)  serum  from  the  infected 
person. 

(1)  Preparation  of  bacterial  emulsion.  In  the  case  of  the 
pyogenic  cocci  a  little  of  a  twenty-four  hour  living  culture  off  a 
sloped  agar  tube  is  taken  and  rubbed  up  in  a  watch-glass  with 
•85  per  cent  saline.  The  mixture  is  placed  in  a  tube  and  centri- 
fugalised  so  as  to  deposit  any  masses  of  bacteria  which  may  be 
present.  Only  by  experience  can  a  knowledge  be  gained  of  the 
amount  of  culture  to  be  used  in  the  first  instance,  but  the 
resultant  emulsion  usually  should  exhibit  only  the  merest  trace  of 
cloudiness  to  the  naked  eye.  Wright  states  it  will  then  contain 
from  7000  to  10,000  million  bacteria  per  c.cm.  If  too  strong  an 
emulsion  be  used  the  leucocytes  may  take  up  so  many  organisms 
that  these  cannot  be  accurately  enumerated.  In  the  case  of 


PREPARATION   OF   THE   SERA  113 

the  tubercle  bacillus  as  short  a  variety  of  the  organism  as 
possible  should  be  selected,  and  a  mass  of  growth  off  a  solid 
medium  is  taken  (bacilli  in  mass  can  be  obtained  in  the  market 
from  wholesale  chemists)  and  is  well  washed  with  changes  of 
distilled  water,  dried  on  filter  paper  in  a  Petri  dish  and 
thoroughly  rubbed  up  with  a  little  1*5  per  cent  saline  in  an 
agate  mortar  so  as  to  disintegrate  the  bacterial  masses  and 
get  an  emulsion  composed  as  far  as  possible  of  individual  bacilli. 
This  must  be  controlled  by  microscopic  examination.  A  thick 
cream  should  be  obtained,  and  this  should  be  sterilised  by 
steaming  for  half  an  hour  on  three  successive  days.  Before 
sterilisation  it  is  convenient  to  seal  up  the  stock  emulsion -in 
small  quantities  in  a  number  of  pieces  of  quill  tubing  so  that  in 
the  subsequent  procedures  only  small  portions  of  the  emulsion 
are  exposed  to  aerial  contamination  at  one  time.  For  actual 
use,  one  of  those  tubes  is  opened,  a  little  is  withdrawn  with  a 
sterile  pipette,  and  a  weak  emulsion  made  in  the  same  way  as 
with  the  staphylococcus  except  that  1'5  per  cent  saline  is  used. 
The  stock  tube  may  be  sealed  with  wax  and  kept  for  use  again. 
A  fresh  emulsion  ought  to  be  made  up  for  each  day's  work. 

(2)  Preparation  of  leucocytes.     Here  the  observer  uses   his 
own  blood  cells.     A  1*5  per  cent  solution  of  sodium  citrate  in 
•85  per  cent  sodium  chloride  is  prepared.     This  is  placed  in  a 
glass  tube  three  inches  long  made  by  drawing  out  a  piece  of 
half-inch  tubing  to  a  point,  the  tube  being  filled  nearly  to  the 
brim.     A  handkerchief  being  bound  round   the  finger,   this  is 
now  pricked  and   the  blood   allowed   to  flow  directly  into  the 
fluid,  to  the  bottom  of  which  it  sinks.     The  tube  ought  to  be 
inverted  between  the  addition  of  every  few  drops  of  blood  so  as 
to  bring  the  blood   in    contact  with  the   citrate  and   prevent 
coagulation.     The  equivalent  of  about  ten  to  twenty  drops  of 
blood  should  be  obtained.      The  diluted  blood  is  then  centri- 
fugalised,  and  when    the    corpuscles   are    separated  the    super- 
natant fluid  is  removed,  '85  per  cent  saline  is  substituted  and 
the  centrifugalisation  repeated.      The  fluid  is  again  removed, 
care  being  taken  not  to  disturb  the  layer  of  white  cells  lying  on 
the  top  of  the  red  corpuscles.     This  layer  is  then  pipetted  off 
into  a  watch-glass  or  tube  and  the  leucocytes  required  are  thus 
obtained. 

(3)  Preparation  of  the  sera.     The  serum  whose  sensitising 
effect  on  the  bacteria  it  is  desired  to  test  is  obtained  by  Wright 
as  follows.     A  "  blood  capsule  "  is  made  by  drawing  a  piece  of 
No.  3  quill  tubing  into  the  shape  shown  in  Fig.  47,  the  part  not 
drawn  out  being  about  one  inch  in  length.     It  is  convenient  to 


114 


MICROSCOPIC   METHODS 


make  a  number  of  these  capsules  at  one  time  and  to  draw  off 
their  extremities  and  seal  them  in  the  flame.  For  use  the  tips  of 
both  extremities  are  broken  off,  the  finger  is  pricked,  and  blood 
allowed  to  pass  into  the  capsule  through  the  bent  limb  till 
the  capsule  is  about  half  full.  The  air  remaining  in  the  capsule 
is  rarefied  by  passing  the  straight  end  through  a  flame  and 
then  sealing  it  off.  By  this  manipulation  the  blood  is  sucked 
over  the  bend  into  the  straight  part  of  the  tube,  and  the  bent 
end  is  now  also  sealed  off  or  closed  with  wax.  It  is  well  to 
shake  the  blood  down  towards  the  closed  straight  end,  care  being 
taken  to  previously  allow  the  glass  to  cool  sufficiently.  The 
capsule  is  now  hung  by  the  bend  on  the  edge  of  a  centrifuge 
tube  'and  the  serum  separated  by  spinning  the  instrument.  In 
any  particular  case  a  capsule  of  serum 
from  the  infected  person  and  one  from 
a  normal  individual  are  prepared. 

The  emulsion,  corpuscles,  and  serum 
being  thus  prepared,  the  next  step  is 
to  mix  them.  This  is  done  by  taking 
a  piece  of  quill  tubing  and  drawing  it 
out  to  a  capillary  point  so  as  to  make 
a  pipette  about  eight  inches  long ;  on 
the  thick  end  of  this  a  rubber  teat  is 
fixed,  and  about  one  inch  from  the 
capillary  point  a  mark  is  made  with 

FTG.  47. -Wright's  Blood-cap-    an  oif  PenciL      From  the  watch-glass 

sule  and  method  of  filling  containing  the  separated  leucocytes  a 

same.  portion  is  sucked  up  to  the  mark  and 

then  an  air-bubble  is  allowed  to  pass 

in.  A  similar  portion  of  the  serum  is  drawn  up,  and  then  another 
air-bubble,  and  finally  a  similar  portion  of  the  bacterial  emulsion. 
The  three  droplets  are  carefully  blown  on  to  a  slide  and  are 
thoroughly  mixed  with  one  another  by  being  alternately 
drawn  up  into  the  tube  and  expelled  several  times.  The 
mixture  is  then  drawn  into  the  tube  and  the  end  sealed  off  in  the 
flame.  The  rubber  nipple  is  removed  and  the  tube  placed  in  the 
incubator  at  37°  for  fifteen  minutes.  A  slide  is  now  prepared 
by  rubbing  it  once  or  twice  with  very  fine  emery  paper 
(No.  000)  and  thoroughly  wiping  it.  This  ;  is  a  procedure 
adopted  by  Wright  to  cause  an  evenly  distributed  film  to  be 
made.  The  tube  being  removed  from  the  incubator  and  the 
end  •  broken  off,  its  contents  are  again  mixed  by  expelling 
and  drawing  up  into  the  tube.  A  minute  droplet  is  placed 
on  the  prepared  slide,  and  by  means  of  the  edge  of  the  end 


GENERAL   BACTERIOLOGICAL   DIAGNOSIS     115 

of  another  slide  a  film  is  made  which  is  then  dried  and  is  ready 
for  staining.  Films  containing  staphylococci  are  stained  either 
by  Leishman's  stain  (q.v.)  or  with  carbol-thionin  blue.  In  the 
former  case  no  fixation  is  necessary,  in  the  latter  it  is  usual  to 
fix  in  saturated  perchloride  of  mercury  for  1|  minutes,  wash  in 
water  and  then  stain.  With  tubercle  films  the  following  is  the 
procedure  :  the  film  is  fixed  for  two  minutes  in  perchloride  of 
mercury,  washed  thoroughly,  stained  with  carbol-fuchsin  as 
usual,  decolorised  with  2'5  per  cent  sulphuric  acid,  cleared  with 
4  per  cent  acetic  acid,  counterstained  with  watery  solution  of 
methylene-blue,  and  dried. 

In  applying  the  technique  two  preparations  are  made,  in  both 
of  which  the  same  emulsion  and  the  same  leucocytes  are 
employed,  but  in  one  of  which  the  bacteria  have  been  exposed 
to  the  serum  of  the  infected  individual  under  observation,  and 
in  the  other  to  that  of  a  normal  person — usually  the  observer 
himself.  Each  of  these  is  now  examined  microscopically  with 
a  movable  stage,  the  number  of  bacteria  in  the  protoplasm  of 
at  least  50  polymorphonucleated  leucocytes  is  counted  and  an 
average  per  leucocyte  struck ;  the  proportion  which  this  average 
in  the  case  of  the  abnormal  serum  bears  to  the  average  in  the 
preparation  in  which  the  healthy  serum  was  used,  constitutes  the 
opsonic  index, — that  of  the  healthy  serum  being  reckoned  as 
unity.  The  reliability  of  the  method  of  course  depends  on  the 
phagocytic  activity  of  the  50  cells  counted  representing  the 
phagocytic  activity  of  all  the  cells  in  the  preparation. 

GENERAL  BACTEEIOLOGICAL  DIAGNOSIS. 

Under  this  heading  we  have  to  consider  the  general  routine 
which  is  to  be  observed  by  the  bacteriologist  when  any  material 
is  submitted  to  him  for  examination.  The  object  of  such 
examination  may  be  to  determine  whether  any  organisms  are 
present,  and  if  so,  what  organisms ;  or  the  bacteriologist  may 
simply  be  asked  whether  a  particular  organism  is  or  is  not 
present.  In  any  case  his  inquiry  must  consist  (1)  of  a  micro- 
scopic examination  of  the  material  submitted ;  (2)  of  an  attempt 
to  isolate  the  organisms  present ;  and  (3)  of  the  identification  of 
the  organisms  isolated.  We  must,  however,  before  considering 
these  points  look  at  a  matter  often  neglected  by  those  who  seek 
a  bacteriological  opinion,  viz.  :  the  proper  methods  of  obtaining 
and  transferring  to  the  bacteriologist  the  material  which  he  is  to 
be  asked  to  examine.  The  general  principles  here  are  (1)  that 
every  precaution  must  be  adopted  to  prevent  the  material  from 


116     GENERAL   BACTERIOLOGICAL   DIAGNOSIS 


being  contaminated  with  extraneous  organisms ;  (2)  that  nothing 
be  done  which  may  kill  any  organisms  which  may  be  proper  to 
the  inquiry ;  and  (3)  that  the  bacteriologist  obtain  the  material 
as  soon  as  possible  after  it  has  been  removed  from  its  natural 
surroundings. 

The  sources  of  materials  to  be  examined,  even  in  patho- 
logical bacteriology  alone,  are  of  course  so  varied  that  we  can 
but  mention  a  few  examples.  It  is,  for  instance,  often  necessary 
to  examine  the  contents  of  an  abscess.  Here  the  skin  must  be 
carefully  purified  by  the  usual  surgical  methods ;  the  knife  used 
for  the  incision  is  preferably  to  be  sterilised  by  boiling,  the  first 
part  of  the  pus  which  escapes  allowed  to  flow  away  (as  it  might 
be  spoiled  by  containing  some  of  the  antiseptics  used  in  the 
purification)  and  a  little  of  what  subsequently  escapes  allowed 
to  flow  into  a  sterile  test-tube.  If  test-tubes 
sterilised  in  a  laboratory  are  not  at  hand,  an 
ordinary  test-tube  may  be  a  quarter  filled  with 
water,  which  is  then  well  boiled  over  a  spirit- 
lamp.  The  tube  is  then  emptied  and  plugged 
with  a  plug  of  cotton  wool,  the  outside  of  which 
has  been  singed  in  a  flame.  Small  stoppered 
bottles  may  be  sterilised  and  used  in  the  same 
way.  A  discharge  to  be  examined  may  be  so 
small  in  quantity  as  to  make  the  procedure 
described  impracticable.  It  may  be  caught  on 
a  piece  of  sterile  plain  gauze,  or  of  plain  ab- 
sorbent wool,  which  is  then  placed  in  a  sterile 
vessel.  Wool  or  gauze  used  for  this  purpose, 
or  for  swobbing  out,  say  the  throat,  to  obtain 
shreds  of  suspicious  matter,  must  have  no 
antiseptic  impregnated  in  it,  as  the  latter  may 
kill  the  bacteria  present  and  make  the  obtaining 
of  cultures  impossible. 

Fluids  from  the  body  cavities,  urine,  etc., 
may  be  secured  with  sterile  pipettes.  To  make 
one  of  these,  take  nine  inches  of  ordinary  quill 
glass-tubing,  draw  out  one  end  to  a  capillary 
diameter,  and  place  a  little  plug  of  cotton  wool 
Insert  this  tube  through  the  cotton  plug  of  an 
ordinary  test-tube  and  sterilise  by  heat.  To  use  it,  remove 
test-tube  plug  with  the  quill  tube  in  its  centre,  suck  up  some 
of  the  fluid  into  the  latter,  and  replace  in  its  former  position 
in  the  test-tube  (Fig.  48).  Another  method  very  convenient  for 
transport  is  to'  make  two  constrictions  on  the  glass  tube  at 


FIG.  48.— Test-tube 
and    pipette    ar- 


ing  bacteria. 
in  the  other  end. 


ROUTINE   EXAMINATION   OF   MATERIAL      117 

suitable  distances,  according  to  the  amount  of  fluid  to  be  taken. 
The  fluid  is  drawn  up  into  the  part  between  the  constrictions, 
but  so  as  not  to  fill  it  completely.  The  tube  is  then  broken 
through  at  both  constrictions  and  the  thin  ends  are  sealed  by 
heating  in  a  flame. 

Solid  organs  to  be  examined  should,  if  possible,  be  obtained 
whole.  They  may  be  treated  in  one  of  two  ways.  1.  The 
surface  over  one  part  about  an  inch  broad  is  seared  with  a 
cautery  heated  to  dull  red  heat.  All  superficial  organisms  are 
thus  killed.  An  incision  is  made  in  this  seared  zone  with  a 
sterile  scalpel,  and  small  quantities  of  the  juice  are  removed  by 
a  platinum  spud  to  make  cover-glass  preparations  and  plate  or 
smear  cultures.  2.  An  alternative  method  is  as  follows  : — The 
surface  is  sterilised  by  soaking  it  well  with  1  to  1000  corrosive 
sublimate  for  half  an  hour.  It  is  then  dried,  and  the  capsule  of 
the  organ  is  cut  through  with  a  sterile  knife,  the  incision  being 
further  deepened  by  tearing.  In  this  way  a  perfectly  uncon- 
taminated  surface  is  obtained.  Hints  are  often  obtained  from 
the  clinical  history  of  the  case  as  to  what  the  procedure  ought 
to  be  in  examination.  Thus,  as  a  matter  of  practice,  cultures 
of  tubercle  and  often  of  glanders  bacilli  can  be  easily  obtained 
only  by  inoculation  experiments.  Typhoid  bacilli  need  hardly 
be  looked  for  in  the  faeces  after  the  first  ten  days  of  the  disease, 
and  so  on. 

Eoutine  Procedure  in  Bacteriological  Examination  of 
Material. — In  the  case  of  a  discharge  regarding  which  nothing  is 
known  the  following  procedure  should  be  adopted  : — (1)  Several 
cover-glass  preparations  should  be  made.  One  ought  to  be 
stained  with  saturated  watery  methylene-blue,  one  with  a  stain 
containing  a  mordant  such  as  Ziehl-Neelsen  carbol-fuchsin,  one 
by  Gram's  method.  (2)  (a)  Gelatin  plates  should  be  made  and 
kept  at  room  temperature,  (b)  a  series  of  agar  plates  or  successive 
strokes  on  agar  tubes  (p.  55)  should  be  made  and  incubated  at 
37°  C.  Method  (b)  of  course  gives  results  more  quickly.  If 
microscopic  investigation  reveals  the  presence  of  bacteria,  it  is 
well  to  keep  the  material  in  a  cool  place  till  next  day  when,  if 
no  growth  has  appeared  in  the  incubated  agar,  some  other  culture 
medium  (e.g.  blood  serum  or  agar  smeared  with  blood)  may  be 
employed.  If  growth  has  "taken  place,  say  in  the  agar  plates, 
one  with  about  200  or  fewer  colonies  should  be  made  the  chief 
basis  for  research.  In  such  a  plate  the  first  question  to  be 
cleared  up  is  :  Do  all  the  colonies  present  consist  of  the  same 
bacterium '?  The  shape  of  the  colony,  its  size,  the  appearance  of 
the  margin,  the  graining  of  the  substance,  its  colour,  etc.,  are  all 


118     GENERAL   BACTERIOLOGICAL   DIAGNOSIS 

to  be  noted.  One  precaution  is  necessary,  viz.,  it  must  be  noted 
whether  the  colony  is  on  the  surface  of  the  medium  or  in  its 
substance,  as  colonies  of  the  same  bacterium  may  exhibit 
differences  according  to  their  position.  The  arrangement  of  the 
bacteria  in  a  surface  colony  may  be  still  more  minutely  studied 
by  means  of  impression  preparations.  A  cover-glass  is  carefully 
cleaned  and  sterilised  by  passing  quickly  several  times  through 
a  Bunsen  flame.  It  is  then  placed  on  the  surface  of  the  medium 
and  gently  pressed  down  on  the  colony.  The  edge  is  then  raised 
by  a  sterile  needle,  it  is  seized  with  forceps,  dried  high  over  the 
flame,  and  treated  as  an  ordinary  cover-glass  preparation.  In 
this  way  very  characteristic  appearances  may  sometimes  be  noted 
and  preserved,  as  in  the  case  of  the  anthrax  bacillus.  The 
colonies  on  a  plate  having  been  classified,  a  microscopic  examina- 
tion of  each  group  may  be  made  by  means  of  cover-glass 
preparations,  and  tubes  of  gelatin  and  agar  are  inoculated  from 
each  representative  colony.  Each  of  the  colonies  used  must  be 
marked  for  future  reference,  preferably  by  drawing  a  circle 
round  it  on  the  under  surface  of  the  plate  or  capsule  with  one 
of  Faber's  pencils  for  marking  on  glass,  a  number  or  letter 
being  added  for  easy  reference. 

The  general  lines  along  which  observation  is  to  be  made  in  the 
case  of  a  particular  bacterium  may  be  indicated  as  follows  : — 

1.  Microscopic  Appearances. — For  ordinary  descriptive  pur- 
poses young  cultures,  say  of  24  hours'  growth,  on  agar  should 
be  used,  though  appearances  in  older  cultures,  such  as  involution 
forms,  etc.,  may  also  require  attention.  Note  (1)  the  form,  (2) 
the  size,  (3)  the  appearance  of  the  protoplasmic  contents, 
especially  as  regards  uniformity  or  irregularity  of  staining,  (4) 
the  method  of  grouping,  (5)  the  staining  reactions.  Has  it 
a  capsule?  Does  the  bacterium  stain  with  simple  watery 
solutions'?  Does  it  require  the  use  of  stains  containing 
mordants  1  How  does  it  behave  towards  Gram's  method  ?  It  is 
important  to  investigate  the  first  four  points  both  when  the 
organism  is  in  the  fluids  or  tissues  of  the  body  and  when  growing 
in  artificial  media,  as  slight  variations  occur.  It  must  also 
be  borne  in  mind  that  slight  variations  are  observed  according 
to  the  kind  and  consistence  of  the  medium  in  which  the  organism 
is  growing.  (6)  Is  it  motile  and  has  it  flagella  ?  If  so,  how  are 
they  arranged  1  (7)  Does  it  form  spores,  and  if  so,  under  what 
conditions  as  to  temperature,  etc.  1 

2.  Growth  Characteristics. — Here  the  most  important  points 
on  which  information  is  to  be  asked  are,  What  are  the 
characters  of  growth  and  what  are  the  relations  of  growth  (1)  to 


GROWTH   CHARACTERISTICS  119 

temperature,  (2)  to  oxygen  1     These  can  be  answered  from  some 
of  the  following  experiments  : — 

A.  Growth  on  gelatin.     (1)  Stab  culture.     Note  (a)  rate  of 
growth ;  (6)  form  of  growth,  (a)  on  surface,  (/?)  in  substance ;  (c) 
presence  or  absence  of  liquefaction  •  (d)  colour ;  (e)  presence  or 
absence  of  gas  formation  and  of  characteristic  smell ;  (/)  relation 
to  reaction  of  medium.     (2)  Streak  culture.     (3)  Shake  culture. 
(4)  Plate  cultures.     Note  appearances  of  colonies  (a)  superficial, 
(6)  deep.     (5)  Growth  in  fluid  gelatin  at  37°  C. 

B.  Growth  on  agar  at  37°  C.     (1)  Stab.     (2)  Streak.     Also 
on  glycerin  agar,  blood  agar,  etc.     Appearances  of  colonies  in 
agar  plates. 

C.  Growth  in  bouillon,  (a)  character  of  growth,  (b)  smell,  (c) 
reaction. 

D.  Growth  on  special  media.      (1)  Solidified  blood  serum. 
(2)  Potatoes.    (3)  Lactose  and  other  sugar  media.    Does  fermenta- 
tion occur  and  is  gas  formed  1    (4)  Milk.    Is  it  curdled  or  turned 
sour  ?    (5)  Litmus  media.    Note  changes  in  colour.    (6)  Peptone 
solution.     Is  indol  formed  ? 

E.  What  is  the  viability  of  organism  on  artificial  media  ? 
3.  Results  of  inoculation  experiments  on  animals. 

By  attention  to  such  points  as  these  a  considerable  knowledge 
is  attained  regarding  the  bacterium,  which  will  lead  to  its 
identification.  In  the  case  of  many  well-known  organisms,  how- 
ever, a  few  of  the  above  points  taken  together  will  often  be 
sufficient  for  the  recognition  of  the  species,  and  experience 
teaches  what  are  the  essential  points  as  regards  any  individual 
organism.  In  the  course  of  the  systematic  description  of  the 
pathogenic  organisms,  it  will  be  found  that  all  the  above  points 
will  be  referred  to,  though  not  in  every  case. 

The  methods  by  which  the  morphological  and  biological  characteristics 
of  any  growth  may  be  observed  have  already  been  fully  described.  It 
need  only  be  pointed  out  here  that  in  giving  descriptions  of  bacteria  the 
greatest  care  must  be  taken  to  state  every  detail  of  investigation.  Thus 
in  any  description  of  microscopic  appearances  the  age  of  the  growth  from 
which  the  preparation  was  made,  the  medium  employed,  the  temperature 
at  which  development  took  place  must  be  noted,  along  with  the  stain 
which  was  used  ;  and  with  regard  to  the  latter  it  is  always  preferable  to 
employ  one  of  the  well-known  staining  combinations,  such  as  Loffler's 
methylene-blue.  Especial  care  is  necessary  in  stating  the  size  of  a 
bacterium.  The  apparent  size  often  shows  slight  variations  dependent 
on  the  stain  used  and  the  growth  conditions  of  the  culture.  Accurate 
measurements  of  bacteria  can  only  be  made  by  preparing  microphoto- 
graphs  of  a  definite  magnification  and  measuring  the  sizes  on  the 
negatives.  From  these  the  actual  sizes  can  easily  be  calculated.  In 
describing  bacterial  cultures  it  must  be  borne  in  mind  that  the  appearances 


120  INOCULATION   OF   ANIMALS 

often  vary  with  the  age.  It  is  suggested  that  in  the  case  of  cultures 
grown  at  from  36°  to  37°  C.  the  appearances  between  24  and  48  hours 
should  be  made  the  basis  of  description,  and  in  the  case  of  cultures 
grown  between  18°  and  22°  C.  the  appearances  between  48  and  72  hours 
should  be  employed.  The  culture  fluids  used  must  be  made  up  and 
neutralised  by  the  precise  methods  already  described.  The  investigator 
must  give  every  detail  of  the  methods  he  has  employed  in  order  that  his 
observations  may  be  capable  of  repetition. 

INOCULATION  OF  ANIMALS.* 

The  animals'generally  chosen  for  inoculation  are  the  mouse, 
the  rat,  the  guinea-pig,  the  rabbit,  and  the  pigeon.  Great  caution 
must  be  shown  in  drawing  conclusions  from  isolated  experiments 
on  rabbits,  as  these  animals  often  manifest  exceptional  symptoms, 
and  are  very  easily  killed.  Dogs  are,  as  a  rule,  rather  insusceptible 
to  microbic  disease,  and  the  larger  animals  are  too  expensive  for 
ordinary  laboratory  purposes.  In  the  case  of  the  mouse  and  rat 
the  variety  must  be  carefully  noted,  as  there  are  differences  in 
susceptibility  between  the  wild  and  tame  varieties,  and  between 
the  white  and  brown  varieties  of  the  latter.  In  the  case  of  the 
wild  varieties,  these  must  be  kept  in  the  laboratory  for  a  week  or 
two  before  use,  as  in  captivity  they  are  apt  to  die  from  very  slight 
causes,  and,  further,  each  individual  should  be  kept  in  a  separate 
cage,  as  they  show  great  tendencies  to  cannibalism.  Of  all  the 
ordinary  animals  the  most  susceptible  to  microbic  disease  is  the 
guinea-pig.  Practically  all  inoculations  are  performed  by  means 
of  the  hypodermic  syringe.  The  best  variety  is  made  on  the 
ordinary  model  with  metal  mountings,  asbestos  washers,  and 
preferably  furnished  with  platinum-iridium  needles.  Before  use 
the  syringe  and  the  needle  are  sterilised  by  boiling  for  five  minutes. 
The  materials  used  for  inoculation  are  cultures,  animal  exudations, 
or  the  juice  of  organs.  If  the  bacteria  already  exist  in  a  fluid 
there  is  no  difficulty.  The  syringe  is  most  conveniently  filled  out 
of  a  shallow  conical  test-glass  which  ought  previously  to  have 
been  covered  with  a  cover  of  filter  paper  and  sterilised.  If  an 
inoculation  is  to  be  made  from  organisms  growing  on  the  surface 
of  a  solid  medium,  either  a  little  ought  to  be  scraped  off  and 
shaken  up  in  sterile  distilled  water  or  '85  per  cent  salt  solution 
to  make  an  emulsion,  or  a  little  sterile  fluid  is  poured  on  the  growth 
and  the  latter  scraped  off  into  it.  This  fluid  is  then  filtered  into 
the  test-glass  through  a  plug  of  sterile  glass  wool.  This  is  easily 
effected  by  taking  a  piece  of  f  in.  glass-tubing  3  in.  long,  drawing 

1  Experiments  on  animals,  of  course,  caunot  be  performed  in  this  country 
without  a  license  granted  by  the  Home  Secretary. 


INOCULATION   OF   ANIMALS 


121 


one  end  out  to  a  fairly  narrow  point,  plugging  the  tube  with 
glass  wool  above  the  point  where  the  narrowing  commences,  and 
sterilising  by  heat.  By  filtering  an  emulsion  through  such  a 
pipette,  flocculi  which  might  block  the  needle  are  removed.  If 
a  solid  organ  or  an  old  culture  is  used  for  inoculation  it  ought 
to  be  rubbed  up  in  a  sterile  porcelain  or  metal  crucible  with  a 
little  sterile  distilled  water,  by  means  of  a  sterile  glass  rod,  and 
the  emulsion  filtered  as  in  the  last  case. 

The  methods  of  inoculation  generally  used  are  :  (1)  by 
scarification  of  the  skin ;  (2)  by  subcutaneous  injection  ;  (3)  by 
intraperitoneal  injection ;  (4)  by  intravenous  injection  ;  (5)  by 
injections  into  special  regions,  such  as  the  anterior  chamber  of  the 
eye,  the  substance  of  the  lung,  etc.  Of  these  (2)  and  (3)  are 
most  frequently  used.  When  an  anaesthetic  is  to  be  administered, 
this  is  conveniently  done  by  placing  the  animal,  along  with  a 
piece  of  cotton-wool  or  sponge  soaked  in  chloroform,  under  a 
bell-jar  or  inverted  glass  beaker  of  suitable  size. 

1.  Scarification. — A  few  parallel   scratches  are  made  in  the 
skin  of  the  abdomen  previously  cleansed,  just  sufficiently  deep 
to  draw  blood,  and  the  infective  material  is  rubbed  in  with  a 
platinum  eyelet.     The  disadvantage  of  this  method  is  that  the 
inoculation    is    easily    contaminated.       The    method    is    only 
occasionally  used. 

2.  Subcutaneous  Injection. — A  hypodermic  syringe  is  charged 
with  the  fluid  to  be  inoculated.     The  hair  is  cut 

off  the  part  to  be  inoculated,  and  the  skin 
purified  with  1  to  1000  corrosive  sublimate. 
The  skin  is  then  pinched  up  and,  the  needle 
being  inserted,  the  requisite  dose  is  administered. 
The  wound  is  then  sealed  with  a  little  collodion. 

3.  Intraperitoneal   Injection. — This    may  be 
performed  by  means  of  a  special  form  of  needle. 
The  needle  is  curved,  and  has  its  opening  not  at 
the  point,  but  in  the  side  in  the  middle  of  the 
arch  (Fig.  49).      The  hair  over  the  lower  part 
of  the  abdomen  is  cut,  and  the  skin  purified  with 
an  antiseptic.     The  whole  thickness  of  the  ab- 
dominal walls  is  then  pinched  up  by  an  assistant, 
between  the  forefingers  and  thumbs  of  the  two 
hands.      The   needle  is    then   plunged   through 
the  fold  thus  formed.      The  result  is  that  the 
hole  in  the    side    of   the    needle  is  within  the 

abdominal    cavity,    and   the    inoculation    can    thus    be    made. 
Intraperitoneal    inoculation    can    also    be    practised    with    an 


FIG.  49.— Hollow 
needle  with 
lateral  aperture 
(at  a)  for  iutra- 
peritoneal  in- 
oculations. 


122  INOCULATION   OF  ANIMALS 

ordinary  needle.  The  mode  of  procedure  is  similar,  but  after 
the  needle  is  plunged  through  the  abdominal  fold,  it  is  partially 
withdrawn  till  the  point  is  felt  to  be  free  in  the  peritoneal 
cavity  when  the  injection  is  made.  There  is  little  risk  of 
injuring  the  intestines  by  either  method. 

4.  Intravenous  Injection. — The  vein  most  usually  chosen  is 
one  of  the  auricular  veins.     The  part  has  the  hair  removed,  the 
skin  is  purified,  and  the  vein  made  prominent  by  pressing  on  it 
between  the  point  of  inoculation  and  the  heart.     The  needle  is 
then  plunged  into  the  vein  arid  the  fluid  injected.     That  it  has 
perforated  the  vessel  will  be  shown  by  the  escape  of  a  little  blood ; 
and  that  the  injection  has  taken  place  into  the  lumen  of  the 
vessel  will  be  known  by  the  absence  of  the  small  swelling  which 
occurs  in  subcutaneous  injections.     If  preferred,  the  vein  may  be 
first  laid  bare  by  snipping  the  skin  over  it.     The  needle  is  then 
introduced. 

5.  Inoculation  into  the  Anterior  Chamber  of  the  Eye, — Local 
anesthesia  is  established  by  applying  a  few  drops  of  2  per  cent 
solution  of   hydrochlorate   of   cocaine.      The   eye   is   fixed  by 
pinching  up  the  orbital  conjunctiva  with  a  pair  of  fine  forceps, 
and  the  edge  of  the  cornea  being  perforated  by  the  hypodermic 
needle,  the  injection  is  easily  accomplished. 

Sometimes  inoculations  are  made  by  planting  small  pieces  of 
pathological  tissues  in  the  subcutaneous  tissue.  This  is  especially 
done  in  the  case  of  glanders  and  tubercle.  The  skin  over  the 
back  is  purified,  and  the  hair  cut.  A  small  incision  is  made  with 
a  sterile  knife,  and  the  skin  being  separated  from  the  subjacent 
tissues  by  means  of  the  ends  of  a  blunt  pair  of  forceps,  a  little 
pocket  is  formed  into  which  a  piece  of  the  suspected  tissue  is 
inserted.  The  wound  is  then  closed  with  a  suture,  and  collodion 
is  applied.  In  the  case  of  guinea-pigs,  the  abdominal  wall  is  to 
be  preferred  as  the  site  of  inoculation,  as  the  skin  over  the  back 
is  extremely  thick. 

Injections  are  sometimes  made  into  other  parts  of  the  body, 
e.g.  the  pleurae  and  the  cranium.  It  is  unnecessary  to  describe 
these,  as  the  application  of  the  general  principles  employed  above, 
together  with  those  of  modern  aseptic  surgery,  will  sufficiently 
guide  the  investigator  as  to  the  technique  which  is  requisite. 

After  inoculation,  the  animals  ought  to  be  kept  in  comfortable 
cages,  which  must  be  capable  of  easy  and  thorough  disinfection 
subsequently.  For  this  purpose  galvanised  iron  wire  cages  are  the 
best.  They  can  easily  be  sterilised  by  boiling  them  in  the  large 
fish-kettle  which  it  is  useful  to  have  in  a  bacteriological  laboratory 
for  such  a  purpose.  It  is  preferable  to  have  the  cages  opening 


COLLODION   CAPSULES  123 

from  above.  Otherwise  material  which  may  be  infective  may  be 
scratched  out  of  the  cage  by  the  animal.  The  general  condition 
of  the  animal  is  to  be  observed,  how  far  it  differs  from  the  normal, 
whether  there  is  increased  rapidity  of  breathing,  etc.  The 
temperature  is  usually  to  be  taken.  This  is  generally  done  per 
rectum.  The  thermometer  (the  ordinary  5  min.  clinical  variety) 
is  smeared  with  vaselin,  and  the  bulb  inserted  just  within  the 
sphincter,  where  it  is  allowed  to  remain  for  a  minute ;  it  is  then 
pushed  well  into  the  rectum  for  five  minutes.  If  this  precaution 
be  not  adopted,  a  reflex  contraction  of  the  vessels  may  take  place, 
which  is  likely  to  vitiate  the  result  by  giving  too  low  a  reading. 

Collodion  Capsules. — These  have  been  used  to  allow  the 
sojourn  of  bacteria  within  the  animal  body  without  their  coming 
into  contact  with  the  cells  of  the  tissues.  Various  substances 
in  solution  can  pass  in  either  direction  through  the  wall  by 
diffusion,  but  the  wall  is  impermeable  alike  to  bacteria  and 
leucocytes.  The  following  method  of  preparing  such  capsules  is 
that  of  M'Rae  modified  by  Harris.  A  gelatine  capsule,  such  as 
is  used  by  veterinary  surgeons,  is  taken,  and  in  one  end  there 
is  fixed  a  small  piece  of  thin  glass  tubing  by  gently  heating  the 
glass  and  inserting  it.  The  tube  becomes  fixed  when  quite  cold, 
and  the  junction  is  then  painted  round  with  collodion,  which 
is  allowed  to  dry  thoroughly.  The  bore  of  the  tubing  is  cleared 
of  any  obstructing  gelatine,  and  the  whole  capsule  is  dipped  into 
a  solution  of  collodion  so  as  to  coat  it  completely.  The  collodion 
is  allowed  to  dry  and  the  coating  is  repeated ;  it  is  also  advis- 
able to  strengthen  the  layer  by  further  painting  it  at  the 
extremity  and  at  the  junction.  The  interior  of  the  capsule  is 
then  filled  with  water  by  a  fine  capillary  pipette,  and  the  capsule 
is  placed  in  hot  water  in  order  to  liquefy  the  gelatine,  which  can 
be  removed  from  the  interior  by  means  of  the  fine  pipette.  The 
sac  is  filled  with  bouillon  and  is  placed  in  a  tube  of  bouillon.  It 
is  then  sterilised  in  the  autoclave.  A  small  quantity  of  the 
bouillon  is  removed,  and  the  contents  are  inoculated  with  the 
particular  bacterium  to  be  studied  or  an  emulsion  of  the  bacterium 
is  added.  The  glass  tubing  is  seized  in  sterile  forceps  and  is 
sealed  off  in  a  small  flame  a  short  distance  above  the  junction. 
The  closed  sac  ought  then  to  be  placed  in  a  tube  of  sterile 
bouillon  to  test  its  impermeability.  The  result  is  satisfactory  if 
no  growth  occurs  in  the  surrounding  medium.  The  sac  with  its 
contents  can  now  be  transferred  to  the  peritoneal  cavity  of  an 
animal. 

Autopsies  on  Animals  dead  or  killed  after  Inoculation. — 
These  should  be  made  as  soon  as  possible  after  death.     It  is 


L24  INOCULATION   OF   ANIMALS 

necessary  to  have  some  shallow  troughs,  constructed  either  of 
metal  or  of  wood  covered  with  metal,  conveniently  with  sheet 
lead,  and  having  a  perforation  at  each  corner  to  admit  a  tape  or 
strong  cord.  The  animal  is  tightly  stretched  out  in  the  trough  and 
tied  in  position.  The  size  of  the  trough  will  therefore  have  to  vary 
with  the  size  of  the  outstretched  body  of  the  animal  to  be  examined. 
In  certain  cases  it  is  well  to  soak  the  surface  of  the  animal  in 
carbolic  acid  solution  (1  to  20)  or  in  corrosive  sublimate  (1  to 
1000)  before  it  is  tied  out.  This  not  only  to  a  certain  extent 
disinfects  the  skin,  but,  what  is  more  important,  prevents  hairs 
which  might  be  affected  with  pathogenic  products  from  getting 
into  the  air  of  the  laboratory.  The  instruments  necessary  are 
scalpels  (preferably  with  metal  handles),  dissecting  forceps,  and 
scissors.  They  are  to  be  sterilised  by  boiling  for  five  minutes. 
This  is  conveniently  done  in  one  of  the  small  portable  sterilisers 
used  by  surgeons.  Two  sets  at  least  ought  to  be  used  in  an 
autopsy,  and  they  may  be  placed,  after  boiling,  on  a  sterile  glass 
plate  covered  by  a  bell-jar.  It  is  also  necessary  to  have  a  medium- 
sized  hatchet-shaped  cautery,  or  other  similar  piece  of  metal.  It 
is  well  to  have  prepared  a  few  freshly-drawn-out  capillary  tubes 
stored  in  a  sterile  cylindrical  glass  vessel,  and  also  some  larger 
sterile  glass  pipettes.  The  hair  of  the  abdomen  of  the  animal  is 
removed.  If  some  of  the  peritoneal  fluid  is  wanted,  a  band 
should  be  cauterised  down  the  linea  alba  from  the  sternum  to  the 
pubes,  and  another  at  right  angles  to  the  upper  end  of  this ;  an 
incision  should  be  made  in  the  middle  of  these  bands,  and  the 
abdominal  walls  thrown  to  each  side.  One  or  more  capillary 
tubes  should  then  be  filled  with  the  fluid  collected  in  the  flanks, 
the  fluid  being  allowed  to  run  up  the  tube  and  the  point  sealed 
off ;  or  a  larger  quantity,  if  desired,  is  taken  in  a  sterile  pipette. 
If  peritoneal  fluid  be  not  wanted,  then  an  incision  may  be  made 
from  the  episternum  to  the  pubes,  and  the  thorax  and  abdomen 
opened  in  the  usual  way.  The  organs  ought  to  be  removed  with 
another  set  of  instruments,  and  it  is  convenient  to  place  them 
pending  examination  in  deep  Petri's  capsules  (sterile).  It  is 
generally  advisable  to  make  cultures  and  film  preparations  from 
the  heart's  blood.  To  do  this,  open  the  pericardium,  sear  the 
front  of  the  right  ventricle  with  a  cautery,  make  an  incision  in  the 
middle  of  the  part  seared,  and  remove  some  of  the  blood  with  a 
capillary  tube  for  future  examination,  or,  introducing  a  platinum 
eyelet,  inoculate  tubes  and  make  cover-glass  preparations  at  once. 
To  examine  any  organ,  sear  the  surface  with  a  cautery,  cut  into  it, 
and  inoculate  tubes  and  make  film  preparations  with  a  platinum 
loop.  For  removing  small  parts  of  organs  for  making  inoculations 


AUTOPSIES   ON   ANIMALS  125 

on  tubes,  a  small  platinum  spud  is  very  useful,  as  the  ordinary 
wires  are  apt  to  become  bent.  Place  pieces  of  the  organs  in 
some  preservative  fluid  for  microscopic  examination.  The  organs 
ought  not  to  be  touched  with  the  ringers.  When  the  examination 
is  concluded  the  body  should  have  corrosive  sublimate  or  carbolic 
acid  solution  poured  over  it,  and  be  forthwith  burned.  The 
dissecting  trough  and  all  the  instruments  ought  to  be  boiled  for 
half  an  hour.  The  amount  of  precaution  to  be  taken  will,  of 
course,  depend  on  the  character  of  the  bacterium  under  investiga- 
tion, but  as  a  general  rule  every  care  should  be  used. 


CHAPTEE   IV. 

BACTERIA  IN   AIR,   SOIL,   AND   WATER. 
ANTISEPTICS. 

IT  is  impossible  here  to  do  more  than  indicate  the  chief  methods 
which  are  employed  by  bacteriologists  in  the  investigation  of  the 
bacteria  present  in  air,  soil,  and  water,  and  to  add  an  outline  of 
the  chief  results  obtained.  In  dealing  with  the  latter  the  subject 
has  been  approached  mainly  from  the  standpoint  of  the  bearings 
which  the  results  have  towards  human  pathology.  In  dealing 
with  antiseptics,  so  far  as  possible  the  effects  of  the  various 
agents  on  the  chief  pathogenic  bacteria  have  been  given,  though 
in  many  cases  our  information  is  very  imperfect. 

AIR. 

Very  little  information  of  value  can  be  obtained  from  the 
examination  of  the  air,  but  the  following  are  the  chief  methods 
used,  along  with  the  results  obtained.  More  can  be  learned 
from  the  examination  of  atmospheres  experimentally  contamin- 
ated than  by  the  investigation  of  the  air  as  it  exists  under 
natural  conditions. 

Methods  of  Examination. — The  -methods  employed  vary  with  the 
objects  in  view.  If  it  be  sought  to  compare  the  relative  richness  of 
different  atmospheres  in  organisms,  and  if  the  atmospheres  in  question 
be  fairly  quiescent,  then  it  is  sufficient  to  expose  gelatin  plates  for 
definite  times  in  the  rooms  to  be  examined.  Bacteria,  or  the  particles  of 
dust  carrying  them,  fall  on  the  plates,  and  from  the  number  of  colonies 
which  develop  a  rough  idea  of  the  richness  of  the  air  in  bacteria  can  be 
obtained.  Petri  states  that  in  five  minutes  the  bacteria  present  in 
10  litres  of  air  are  deposited  on  100  square  centimetres  of  a  gelatin 
plate. 

126 


METHODS   OF   EXAMINATION 


127 


More  complete  results  are  available  when  some  method  is  employed  by 
which  the  bacteria  in  a  given  quantity  of  air  are  examined.  The  oldest 
method  employed,  and  one  which  is  still  used,  is  that  of  Hesse.  The 
apparatus  is  shown  in  Fig.  50.  It  consists  of  a  cylindrical  tube  a  about 
20  inches  long  and  2  inches  in  diameter.  At  one  end  this  is  closed  by  a 
rubber  cork  having  a  piece  of  quill  tubing,  /,  passing  through  it  and 
projecting  some  distance  into  the  interior.  For  use  the  tube  is  sterilised 
in  a  tall  "Koch,"  and  then  a  quantity  of  peptone  gelatin,  sufficient  to 
cover  the  whole  interior  to  the  thickness  of  an  ordinary  gelatin  plate,  is 
poured  in.  This  gelatin 

is  kept  from  escaping  by  ./  ,.« 

the  projection  of  the  quill 
tubing  into  the  lumen  of 
the  large  tube.  A  plug 
of  cotton  wool  is  now 
placed  in  the  outer  end 
of  the  quill  tubing.  Over 
the  other  end  of  the  large 
tube  is  tied  a  sheet  of 
rubber  having  a  hole 
about  a  quarter  of  an  inch 
in  diameter  in  its  centre, 
and  over  this  again  is  tied 
a  piece  of  similar  but  un- 
perforated  sheet  rubber. 
The  tube  is  then  steri- 
lised in  the  tall  " Koch.' 
On  removal  from  this  it 
is  rolled,  after  the  manner 
of  an  Esmarch's  tube 
(q.v.),  till  the  gelatin 
is  set  as  a  layer  over 
its  interior,  and  it  is  then 
placed  horizontally  on 
the  tripod  as  shown.  The 
other  part  of  the  appar- 
atus is  an  aspirator  by 
means  of  which  a  known 
quantity  of  air  can  be 
brought  in  contact  with 
the  gelatin.  It  consists 


FIG.  50. — Hesse's  tube,  mounted  for  use. 


of  two  conical  glass  flasks  connected  by  means  of  a  tube  which  passes 
through  the  cork  of  each  down  to  the  bottom  of  the  flask.  When  this 
tube  is  filled  with  water,  it,  of  course,  can  act  as  a  syphon  tube  between 
volumes  of  water  in  the  flasks.  Such  a  syphon  system  being  established, 
the  levels  of  the  water  are  marked  on  the  flasks,  and  to  one  a  litre  of 
water  is  added,  and  by  depressing  flask  b  the  whole  litre  can  be  got  into 
it  and  the  connecting  tube  c  is  then  clamped.  The  two  flasks  are  then 
connected  by  a  rubber  tube  with  the  tube/,  the  clamp  on  c  is  opened, 
and  the  passing  of  a  litre  of  water  into  d  will  draw  a  litre  of  air  through 
the  gelatin  tube,  when  the  outer  rubber  sheet  is  removed  from  the  end 
and  the  clamp  h  opened.  By  disconnecting  at  g  and  reversing  the  syphon 
flasks,  another  litre  can  be  sucked  through,  and  so  any  desired  quantity 
of  air  can  be  brought  in  contact  with  the  gelatin.  The  speed  ought  not 
to  be  more  than  one  litre  in  two  minutes,  and  in  such  a  case  practically 


128 


BACTERIA   IN  AIR 


all  the  organisms  will  be  found  to  have  fallen  out  of  the  air  on  to  the 
gelatin  in  the  course  of  their  transit.  This  fact  can  be  tested  by 
interposing  between  the  tube  a  and  the  aspirator  a  second  tube  prepared 
in  the  same  way,  which  ought,  of  course,  to  show  no  growth.  When 
forty-eight  hours  at  20°  C.  or  four  days  at  lower  temperature  have 
elapsed,  the  colonies  which  develop  in  a  may  be  counted.  The  dis- 
advantage of  the  method  is  that  if  particles  of  dust  carrying  more  than 
one  bacterium  alight  on  the  gelatin,  these  bacteria  develop  in  one  colony, 
and  thus  the  enumeration  results  may  be  too  low  ;  difficulties  may  also 
arise  from  liquefying  colonies  developing  in  the  upper  parts  of  the  tube 
and  running  over  the  gelatin. 

Petri's  Sand-Filter  Method. — A  glass  tube  open  at  both  ends,  and 
about  3^  inches  long  and  half  an  inch  wide,  is  taken,  and  in  its  centre  is 
placed  a  transverse  diaphragm  of  very  fine  iron 
gauze  (Fig.  51,  e]  ;  on  each  side  of  this  is  placed 
some  fine  quartz  sand  which  has  been  well  washed, 
dried,  and  burned  to  remove  all  impurities,  and  this 
is  kept  in  position  by  cotton  plugs.  The  whole  is 
sterilised  by  dry  heat.  One  plug  is  removed  and  a 
sterile  rubber  cork,  c,  inserted,  through  which  a  tube, 
d,  passes  to  an  exhausting  apparatus.  The  tube  is 
then  clamped  in  an  upright  position  in  the  atmo- 
sphere to  be  examined,  with  the  remaining  plug, 
/,  uppermost.  The  latter  is  removed  and  the  air 
sucked  through.  Difficulty  may  be  experienced 
from  the  resistance  of  the  sand  if  quick  nitration  be 
attempted.  The  best  means  to  adopt  is  to  use  an 
air-pump — the  amount  of  air  drawn  per  stroke  of 
which  is  accurately  known — and  have  a  manometer 
(as  in  Fig.  31)  interposed  between  the  tube  and  the 
pump.  Between  each  two  strokes  of  the  air-pump 
the  mercury  is  allowed  to  return  to  zero.  After 
the  required  amount  of  air  has  passed,  the  sand  a 
is  removed,  and  is  distributed  among  a  number  of 
sterile  gelatin  tubes  which  are  well  shaken  ;  plate 
cultures  are  then  made,  and  when  growth  has  occurred 
the  colonies  are  enumerated  ;  the  sand  b  is  similarly 
treated  and  acts  as  a  control. 

When  it  is  necessary  to  examine  air  for  particular 
organisms,  special  methods  must  often  be  adopted. 
Thus  in  the  case  of  the  suspected  presence  of  tubercle  bacilli  a  given 
quantity  of  air  is  drawn  through  a  small  quantity  of  water  and  then 
injected'  into  a  guinea-pig. 

It  must  be  admitted  that  comparatively  little  information 
bearing  on  the  harmlessness  or  harmfulness  of  the  air  is  obtain- 
able by  the  mere  enumeration  of  the  living  organisms  present, 
for  under  certain  conditions  the  number  may  be  increased  by 
the  presence  of  many  individuals  of  a  purely  non-pathogenic 
character.  The  organisms  found  in  the  air  belong  to  two 
groups — firstly,  a  great  variety  of  bacteria  ;  secondly,  yeasts  and 
the  spores  of  moulds  and  of  the  lower  fungi.  With  regard  to 
the  spores,  the  organisms  from  which  they  are  derived  often 


FIG.  51. — Petri's 
sand  filter. 


PETRI'S   SAND-FILTER   METHOD  129 

consist  of  felted  masses  of  threads,  from  which  are  thrust  into  the 
air  special  filaments,  and  in  connection  with  these  the  spores  are 
formed.  By  currents  of  air  these  latter  can  easily  be  detached, 
and  may  float  about  in  a  free  condition.  With  the  bacteria,  on 
the  other  hand,  the  case  is  different.  Usually  these  are  growing 
together  in  little  masses  on  organic  materials,  or  in  fluids,  and 
it  is  very  much  by  the  detachment  of  minute  particles  of  the 
substratum  that  the  organisms  become  free.  The  entrance  of 
bacteria  into  the  air,  therefore,  is  associated  with  conditions 
which  favour  the  presence  of  dust,  minute  droplets  of  fluid,  etc. 
The  presence  of  dust  in  particular  would  specially  favour  a  large 
number  of  bacteria  being  observed,  and  this  is  the  case  with  the 
air  in  many  industrial  conditions,  where  the  bacteria,  though 
numerous,  may  be  quite  innocuous.  Great  numbers  of  bacteria 
thus  may  not  indicate  any  condition  likely  to  injure  health,  and 
this  may  be  true  also  even  when  the  bacteria  come  from  the 
crowding  together  of  a  number  of  healthy  human  beings.  On 
the  other  hand,  there  is  no  doubt  that  disease  germs  can  be 
disseminated  by  means  of  the  air.  The  possibility  of  this 
has  been  shown  experimentally  by  infecting  the  mouth  with  the 
b.  prodigiosus,  which  is  easily  recognised  by  its  brilliantly 
coloured  colonies,  and  then  studying  its  subsequent  distribution. 
Most  important  here  is  the  infection  of  the  air  from  sick  persons. 
The  actions  of  coughing,  sneezing,  speaking,  and  even  of  deep 
breathing,  distribute,  often  to  a  considerable  distance,  minute 
droplets  of  secretions  from  the  mouth,  throat,  and  nose,  and  these 
may  float  in  the  air  for  a  considerable  time.  Even  five  hours 
after  an  atmosphere  has  been  thus  infected  evidence  may  be 
found  of  bacteria  still  floating  free.  Before  this  time,  however, 
most  of  the  bacteria  have  settled  upon  various  objects,  where 
they  rapidly  dry,  and  are  no  longer  displaceable  by  ordinary  air 
currents.  The  diseases  of  known  etiology  where  infection  can 
thus  take  place  are  diphtheria,  influenza,  pneumonia,  and  phthisis  ; 
and  here  also  probably  whooping-cough,  typhus  fever,  and  measles 
are  to  be  added,  though  the  morbific  agents  are  unknown.  In 
the  case  of  phthisis,  the  alighting  of  tubercle  bacilli  has  been 
demonstrated  on  cover-glasses  held  before  the  mouths  of  patients 
while  talking,  and  animals  made  to  breathe  directly  in  front  of 
the  mouths  of  such  patients  have  become  infected  with 
tuberculosis.  Apart  from  direct  infection  from  individuals, 
however,  pathogenic  bacteria  may  be  spread  in  some  cases  from 
the  splashing  of  infected  water,  as  from  a  sewage  outfall. 
This  possibility  has  to  be  recognised  especially  in  the  cases  of 
typhoid  and  cholera.  Besides  infection  through  fluid  particles, 
9 


130  BACTERIA  IN   AIR 

infection  can  be  caused  in  the  air  by  dust  coming,  say,  from 
infected  skin  or  clothes,  etc.  Fliigge,  in  dealing  with  this 
subject  in  an  experimental  inquiry,  distinguishes  between  large 
particles  of  dust  which  require  an  air  current  moving  at  the  rate 
of  1  centimetre  per  second  to  keep  them  suspended,  and  the  finer 
dust  which  can  be  kept  in  suspension  by  currents  moving  at  from 
1  to  4  millimetres  per  second.  In  the  former  case,  when  once 
the  particles  alight  they  cannot  be  displaced  by  currents  of  air 
except  when  these  are  moving  at,  at  least,  5  metres  per  second, 
but  the  brushing,  shaking,  or  beating  of  objects  may,  of  course, 
distribute  them.  In  the  case  of  the  finer  dust  the  particles  will 
remain  for  long  suspended,  and  when  they  have  settled  can  be 
more  easily  displaced,  as  by  the  waving  of  an  arm,  breathing, 
etc.  With  regard  to  infection  by  dust,  a  most  important  factor, 
however,  is  whether  or  not  the  infecting  agent  can  preserve  its 
vitality  in  a  dry  condition.  In  the  case  of  a  sporing  organism 
such  as  anthrax,  vitality  is  preserved  for  long  periods  of  time, 
and  great  resistance  to  drying  is  also  possessed  by  the  tubercle 
and  diphtheria  bacilli ;  but  apart  from  such  cases  there  is  little 
doubt  that  infection  is  usually  necessarily  associated  with  the 
transport  of  moist  particles,  and  is  thus  confined  to  a  limited 
area  around  a  sick  person.  Among  diseases  which  may 
occasionally  be  thus  spread  cholera  and  typhoid  have  been  classed. 
Considerable  controversy  has  arisen  with  regard  to  certain  out- 
breaks of  the  latter  disease,  which  have  apparently  been  spread 
by  dusty  winds,  although  we  have  the  fact  that  the  typhoid 
bacillus  does  not  survive  being  dried  even  for  a  short  time. 
It  appears,  however,  that  in  such  epidemics  the  transport  of 
infection  by  .means  of  insects  carried  by  the  wind  has  not  been 
entirely  excluded. 

As  in  the  cases  of  the  soil  and  of  water,  presently  to  be  described, 
attempts  have  been  made  to  obtain  indirect  evidence  of  the  contamination 
of  the  air  by  man.  Thus  Gordon  has  shown  that  certain  streptococci 
are  common  in  the  saliva  ;  these  resemble  the  streptococcus  pyogenes,  but 
are  relatively  non-pathogenic,  grow  well  at  37°  C.  and  under  anaerobic 
conditions,  cause  clotting  and  acid-formation  in  litmus  milk  at  37°,  and 
in  neutral-red  media  have  an  action  resembling  that  of  b.  coli.  These 
characters  serve,  according  to  Gordon,  to  differentiate  organisms  of 
human  origin  from  ordinary  streptococci  occurring  in  the  air  and  which 
he  states  grow  better  at  about  22°  C.,  are  facultative  anaerobes  and 
do  not  produce  the  changes  in  milk  and  in  neutral-red  media.  Thus 
the  finding  of  streptococci  of  the  first  group  in  plates  exposed  to  air 
would  indicate  that  a  human  source  was  probable,  and,  if  the  observation 
were  made  on  air  from  the  neighbourhood  of  a  sick  person,  that  risk  of 
the  dissemination  of  disease  germs  was  present.  The  value  of  this  as  a 
practical  method  has  yet  to  be  determined. 


BACTERIA   IN   SOIL  131 

SOIL. 

The  investigation  of  the  bacteria  which  may  be  found  in  the 
soil  is  undertaken  from  various  points  of  view.  Information 
may  be  desired  as  to  the  change  its  composition  undergoes  by  a 
bacterial  action,  the  result  of  which  may  be  an  increase  in 
fertility  and  thus  in  economic  value.  Under  this  head  may  be 
grouped  inquiries  relating  to  the  bacteria  which  convert 
ammonia  and  its  salts  into  nitrates  and  nitrites,  and  to  the 
organisms  concerned  in  the  fixation  of  the  free  nitrogen  of  the 
air.  The  discussion  of  the  questions  involved  in  such  inquiries 
is  outside  the  scope  of  the  present  chapter,  which  is  more  con- 
cerned with  the  relation  of  the  bacteriology  of  the  soil  to  questions 
of  public  health.  So  far  as  this  narrower  view  is  concerned,  soil 
bacteria  are  chiefly  of  importance  in  so  far  as  they  can  be  washed 
out  of  the  soils  into  potable  water  supplies.  An  important  aspect 
of  this  question  thus  is  as  to  the  significance  of  certain  bacterio- 
logical appearances  in  a  water  in  relation  to  the  soil  from  which 
it  has  come  or  over  which  it  has  flowed.  In  this  country  these 
questions  have  been  chiefly  investigated  by  Houston,  and  it  is 
from  his  papers  that  the  following  account  is  largely  taken. 

Methods  of  Examination. — For  examination  of  soil  on  surface  or  not 
far  from  surface,  Houston  recommends  tin  troughs  10  in.  by  3  in.  and 
pointed  at  one  extremity,  to  be  wrapped  in  layers  of  paper  and  sterilised 
by  dry  heat.  If  several  of  these  be  provided,  then  the  soil  can  be  well 
rubbed  Up  and  a  sample  secured  and  placed  in  a  sterile  test-tube  for 
examination  as  soon  as  convenient  after  collection.  If  samples  are  to 
be  taken  at  some  depth  beneath  the  surface,  then  a  special  instrument 
of  which  many  varieties  have  been  devised  must  be  used.  The  general 
form  of  these  is  that  of  a  gigantic  gimlet  stoutly  made  of  steel.  Just 
above  the  point  of  the  instrument  the  shaft  has  in  it  a  hollow  chamber, 
and  a  sliding  lateral  door  in  this  can  be  opened  and  shut  by  a  mechanism 
controlled  at  the  handle.  The  chamber  being  sterilised  and  closed,  the 
instrument  is  bored  to  the  required  depth,  the  door  is  slid  back,  and  by 
varying  devices  it  is  effected  that  the  chamber  is  filled  with  earth  ;  the 
door  is  reclosed  and  the  instrument  withdrawn. 

In  any  soil  the  two  important  lines  of  inquiry  are  first  as  to  the  total 
number  of  organisms  (usually  reckoned  per  gramme  of  the  fresh  sample), 
and  secondly  as  to  the  varieties  of  organisms  present.  The  number  of 
organisms  present  in  a  soil  is  often,  however,  so  enormous  that  it  is  con- 
venient to  submit  only  a  fraction  of  a  gramme  to  examination.  The 
method  employed  is  to  weigh  the  tube  containing  the  soil,  shake  out  an 
amount  of  about  the  size  of  a. bean  into  a  litre  of  distilled  water,  and  re- 
weigh  the  tube.  The  amount  placed  in  the  water  is  distributed  as 
thoroughly  as  possible  by  shaking,  and,  if  necessary,  by  rubbing  down 
with  a  sterile  glass  rod,  and  small  quantities  measured  from  a  graduated 
pipette  are  used  for  the  investigation.  For  estimating  the  total  number  of 
organisms  present  in  the  portion  of  soil  used,  small  quantities,  say  '1  c.c. 
and  1  c.c.,  of  the  fluid  are  added  to  melted  tubes  of  ordinary  alkaline 
peptone  gelatin  ;  after  being  shaken,  the  gelatin  is  plated,  incubated  at 


132  BACTERIA   IN   SOIL 

22°  C.,  and  the  colonies  are  counted  as  late  as  the  liquefaction,  which 
always  occurs  round  some  of  them,  will  allow.  -From  these  numbers  the 
total  number  of  organisms  present  in  the  amount  of  soil  originally  present 
can  be  calculated. 

The  numbers  of  bacteria  in  the  soil  vary  very  much.  Accord- 
ing to  Houston's  results,  fewest  occur  in  uncultivated  sandy  soils, 
these  containing  on  an  average  100,000  per  gramme.  Peaty  soils, 
though  rich  in  organic  matter,  also  give  low  results,  it  being 
possible  that  the  acidity  of  such  soils  inhibits  free  bacterial 
growth.  Garden  soils  yield  usually  about  1,500,000  bacteria 
per  gramme,  but  the  greatest  numbers  are  found  in  soils  which 
have  been  polluted  by  sewage,  when  the  figures  may  rise  to 
115,000,000.  In  addition  to  the  enumeration  of  the  numbers 
of  bacteria  present,  it  is  a  question  whether  something  may  not 
be  gained  from  a  knowledge  of  the  number  of  spores  present  in 
a  soil  relative  to  the  total  number  of  bacteria.  This  is  a  point 
which  demands  further  inquiry,  especially  by  the  periodic  investi- 
gation of  examples  of  different  classes  of  soils.  The  method  is  to 
take  1  c.c.  of  such  a  soil  emulsion  as  that  just  described,  add  it 
to  10  c.c.  of  gelatin,  heat  for  ten  minutes  at  80°  C.  to  destroy 
the  non-spored  bacteria,  plate,  incubate,  and  count  as  before. 

Besides  the  enumeration  of  the  numbers  of  bacteria  present  in 
a  soil,  an  important  question  in  its  bacteriological  examination 
lies  in  inquiring  what  kinds  of  bacteria  are  present  in  any  par- 
ticular case.  Practically  this  resolves  itself  into  studying  the 
most  common  bacteria  present,  for  the  complete  examination  of 
the  bacterial  flora  of  any  one  sample  would  occupy  far  too  much 
time.  Of  these  common  bacteria  the  most  important  are  those 
from  whose  presence  indications  can  be  gathered  of  the  con- 
tamination of  the  soil  by  sewage,  for  from  the  public  health 
standpoint  this  is  by  far  the  most  important  question  on  which 
bacteriology  can  shed  light. 

Bacillus  mycoides. — This  bacillus  is  1  '6  to  2'4  n  in  length  and  about  '9 
in  breadth.  It  grows  in  long  threads  which  often  show  motility.  It 
can  be  readily  stained  by  such  a  combination  as  carbol-thionin,  and  re- 
tains the  dye  in  Gram's  method.  All  ordinary  media  will  support  its 
growth,  and,  in  surface  growths  on  agar  or  potato  spore,  formation  is 
readily  produced.  Its  optimum  temperature  is  about  18°  C.  On  gelatin 
plates  it  shows  a  very  characteristic  appearance.  At  first  under  a  low 
power  it  shows  a  felted  mass  of  filaments  throwing  out  irregular  shoots 
from  the  centre,  and  later  to  the  naked  eye  these  appear  to  be  in  the 
form  of  thick  threads  like  the  growth  of  a  mould.  They  rapidly  spread 
over  the  surface  of  the  medium,  and  the  whole  resembles  a  piece  of  wet 
teased-out  cotton  wool.  The  gelatin  is  liquefied. 

Cladothrices. — Of  these  several  kinds  are  common  in  the  soil.  The 
ordinary  cladothrix  dichotoma  is  among  them.  This  organism  appears 


BACTERIA   IN   SOIL  133 

as  colourless  flocculent  growth  with  an  opaque  centre,  and  can  be  seen 
under  the  microscope  to  send  out  into  the  medium  apparently  branched 
threads  which  vary  in  thickness,  being  sometimes  2  /j.  across.  They 
consist  of  rods  enclosed  in  a  sheath.  These  rods  may  divide  at  any 
point,  and  thus  the  terminal  elements  may  be  pushed  along  the  sheath. 
Sometimes  the  sheath  ruptures,  and  thus  by  the  extrusion  of  these 
dividing  cells  and  their  further  division  the  branching  appearance  is 
originated.  Reproduction  takes  place  by  the  formation  of  gonidia  in  the 
interior  of  the  terminal  cells.  These  gonidia  acquire  at  one  end  a  bundle 
of  flagella,  and  for  some  time  swim  free  before  becoming  attached  and 
forming  a  new  colony.  Houston  describes  as  occurring  in  the  soil  another 
variety,  which  with  similar  microscopic  characters  appears  as  a  brownish 
growth  with  a  pitted  surface,  and  diffusing  a  bismarck-brown  pigment 
into  the  gelatin  which  it  liquefies. 

A  few  experiments  made  with  an  ordinary  field  soil  will,  however, 
familiarise  the  worker  with  the  non-pathogenic  bacteria  usually  present. 
We  have  referred  to  these  two  because  of  their  importance.  In  regard 
to  pathogenic  organisms,  especially  in  relation  to  possible  sewage  con- 
tamination, attention  is  to  be  directed  to  three  groups  of  organisms, 
those  resembling  the  b.  coli,  the  bacillus  enteritidis  sporogenes,  and  the 
streptococcus  pyogenes.  The  characters  of  the  first  two  of  these  will 
be  found  in  the  chapter  on  Typhoid  Fever  ;  of  the  third  in  Chap.  VI. 
For  the  detection  of  these  bacteria  Houston  recommends  the  following 
procedure. 

(a)  The  B.  coli  group.  A  third  of  a  gramme  of  soil  is  added  to  10  c.c. 
phenol  broth  (vide  chapter  on  Typhoid  Fever)  and  incubated  at  37°  C. 
In  this  medium  very  few  if  any  other  bacteria  except  those  of  the  b.  coli 
group  will  grow,  so  that  if  after  twenty-four  hours  a  turbidity  appears, 
some  of  the  latter  may  be  suspected  to  be  present.  In  such  a  case  a 
loopful  of  the  broth  is  shaken  up  in  5  c.c.  sterile  distilled  water,  and  of 
this  one  or  two  loopfuls  are  spread  over  the  surface  of  a  solid  plate  of 
phenol  gelatin  in  a  Petri  capsule  either  by  means  of  the  loop  or  of 
a  small  platinum  spatula,  and  the  plate  is  incubated  at  20°  C.  Any 
colonies  which  resemble  b.  coli  are  then  examined  by  the  culture 
methods  detailed  under  that  organism.  Further,  all  organisms  having 
the  microscopic  appearances  of  b.  coli,  and  which  generally  conform  to 
its  culture  reactions,  are  to  be  reckoned  in  the  coli  group.  The  media 
of  MacConkey  and  Drigalski  are  very  useful  in  connection  with  the  separa- 
tion of  such  soil  organisms  (v.  pp.  42,  43). 

(&)  The  Bacillus  enteritidis  sporogenes.  To  search  for  this  organism  1 
gramme  of  the  soil  is  thoroughly  distributed  in  100  c.c.  sterile  distilled 
water,  and  of  this  1  c.c.,  *1  c.c.,  and  '01  c.e:  is  added  to  each  of  three  sterile 
milk  tubes.  These  are  heated  to  80°  C.  for  ten  minutes  and  then  cultivated 
anaerobically  at  37°  C.  for  twenty-four  hours.  If  the  characteristic  appear- 
ances seen  in  such  cultures  of  the  b.  enteritidis  (q.v.)  are  developed, 
then  it  may  fairly  safely  be  deduced  that  it  is  this  organism  which  has 
produced  them. 

(c)  Faecal  Streptococci.  The  method  here  is  to  pour  out  a  tube  of  agar 
into  a  Petri  capsule,  and  when  it  has  solidified  to  spread  out  "1  c.c.  of 
the  emulsion  of  soil  over  it  and  incubate  at  37°  C.  for  twenty-four  hours. 
At  this  temperature  many  of  the  non-pathogenic  bacteria  grow  with 
difficulty,  and  thus  the  number  of  colonies  which  develop  is  relatively 
small.  Colonies  having  appearances  resembling  those  of  the  streptococcus 
pyogenes  (q.v.)  can  thus  be  investigated.  Much  work  has  been  devoted 
to  the  question  of  these  faecal  streptococci  presenting  specific  characters  by 


134  BACTERIA  IN   SOIL 

which  they  could  be  differentiated  from  other  streptococci.  No  definite 
results  have  as  yet  been  obtained.  Houston  gives  as  the  general 
characters  of  these  organisms  that  they  usually  grow  in  short  chains, 
that  they  produce  uniform  turbidity  in  broth,  that  they  give  rise  to  acid  - 
and  clot  in  litmus  milk  at  37°  C.,  and  that  they  are  non-pathogenic  to 
mice.  The  important  point  is  to  recognise  that  streptococci  of  fairly 
ordinary  types  exist  in  great  numbers  in  human  faeces,  and  that  when 
in  any  circumstances  fsecal  contamination  is  suspected  the  isolation  of 
streptococci  strengthens  the  suspicion. 

We  may  now  give  in  brief  the  results  at  which  Houston  has 
arrived  by  the  application  of  these  methods.  First  of  all,  un- 
cultivated soils  contain  very  few,  if  any,  representatives  of  the 
b.  mycoides,  and  this  is  also  true  to  a  less  extent  of  the 
cladothrices.  Cultivated  soils,  on  the  other  hand,  do  practically 
always  contain  these  organisms.  With  regard  to  the  b.  coli  its 
presence  in  a  soil  must  be  looked  on  as  indicative  of  recent 
pollution  with  excremental  matter.  The  presence  of  b. 
enteritidis  is  also  evidence  of  such  pollution,  but  from  the 
fact  that  it  is  a  sporing  organism  this  pollution  may  not  have 
been  recent.  With  regard  to  the  streptococci,  on  the  other 
hand,  the  opinion  is  advanced  that  their  presence  is,  on  account 
of  their  want  of  viability  outside  the  animal  body,  to  be  looked 
on  as  evidence  of  extremely  recent  excremental  pollution.  The 
very  great  importance  of  these  results  in  relation  to  the 
bacteriological  examination  of  water  supplies  will  be  at  once 
apparent,  and  will  be  referred  .to  again  in  connection  with  the 
subject  of  water. 

While  such  means  have  been  advanced  for  the  obtaining  of 
indirect  evidence  of  excremental  pollution  of  soil,  and  therefore 
of  a  pollution  dangerous  to  health  from  the  possible  presence  of 
pathogenic  organisms  in  excreta,  investigations  have  also  been 
conducted  with  regard  to  the  viability  in  the  soil  of  pathogenic 
bacteria,  especially  of  those  likely  to  be  present  in  excreta,  namely, 
the  typhoid  and  cholera  organisms.  The  solution  of  this  problem 
is  attended  with  difficulty,-  as  it  is  not  easy  to  identify  these 
organisms  when  they  are  present  in  such  bacterial  mixtures  as 
naturally  occur  in  the  soil.  Now  there  is  evidence  that  bacteria 
when  growing  together  often  influence  each  other's  growth  in  an 
unfavourable  way,  so  that  it  is  only  by  studying  the  organisms  in 
question  when  growing  in  unsterilised  soils  that  information  can 
be  obtained  as  to  what  occurs  in  nature.  For  instance,  it  has 
been  found  that  the  b.  typhosus,  when  grown  in  an  organically 
polluted  soil  which  has  been  sterilised,  can  maintain  its  vitality 
for  fifteen  weeks,  but  if  the  conditions  occurring  naturally  be  so 
far  imitated  by  growing  it  in  soil  in  the  presence  of  a  pure  culture 


BACTERIA  IN   WATER  135 

of  one  or  other  certain  soil  bacteria,  it  is  found  that  sometimes  the 
typhoid  bacillus,  sometimes  the  soil  bacterium  in  the  course  of  a 
few  weeks,  or  even  in  a  few  days,  disappears.  Further,  the  char- 
acter of  the  soil  exercises  an  important  effect  on  what  happens ; 
for  instance,  the  typhoid  bacillus  soon  dies  out  in  a  virgin  sandy 
soil,  even  when  it  is  the  only  organism  present.  In  experiments 
made  by  sowing  cultures  of  cholera  and  diphtheria  in  plots  in  a 
field  it  was  found  that  after,  at  the  longest,  forty  days  they  were 
no  longer  recognisable.  Further,  it  is  a  question  whether 
ordinary  disease  organisms,  even  if  they  remain  alive,  can 
multiply  to  any  great  extent  in  soil  under  natural  conditions. 
If  we  are  dealing  with  a  sporing  organism  such  as  the  b. 
anthracis,  the  capacity  for  remaining  in  a  quiescent  condition  of 
potential  pathogenicity  is,  of  course,  much  greater.  The  most 
important  principle  to  be  deduced  from  these  experiments  is  that 
the  ordinary  conditions  of  soil  rather  tend  to  be  unfavourable 
to  the  continued  existence  of  pathogenic  bacteria,  so  that  by 
natural  processes  soil  tends  to  purify  itself.  It  must,  however, 
be  noted  that  such  an  organism  as  the  typhoid  bacillus  can  exist 
long  enough  in  soil  to  be  a  serious  source  of  danger. 

WATER. 

In  the  bacteriological  examination  of  water  three  lines  of 
inquiry  may  have  to  be  followed.  First,  the  number  of  bacteria 
per  cubic  centimetre  may  be  estimated.  Second,  the  kinds  of 
bacteria  present  may  be  investigated.  Third,  it  may  be  necessary 
to  ask  if  a  particular  organism  is  present,  and,  if  so,  in  what 
number  per  c.c.  it  occurs. 

Methods. — In  the  two  first  cases  a  small  quantity  ("5-1  c.c.)  is  taken 
in  a  sterile  pipette  and  added  to  a  tube  of  gelatin,  which  is  then  plated 
and  incubated  at  the  room  temperature.  In  the  case  of  water  taken 
from  a  house  tap  the  water  should  be  allowed  to  run  for  several  hours 
before  the  sample  is  taken,  as  water  standing  in  pipes  in  a  house  is  under 
very  favourable  conditions  for  multiplication  of  bacteria  taking  place, 
and  if  this  precaution  be  not  adopted  an  altogether  erroneous  idea  of  the 
number  present  may  be  obtained.  In  the  case  of  the  examination  of 
river  water  the  gelatin  plates  ought  to  be  prepared  on  the  spot  ;  at  any 
rate,  the  time  elasping  between  the  sample  being  taken  and  the  plates 
being  prepared  must  be  as  short  as  possible,  otherwise  the  bacteria  will 
multiply,  and  again  an  erroneous  idea  of  their  number  be  obtained. 
When  samples  have  to  be  taken  for  transport  to  the  laboratory,  these 
are  best  collected  in  four-ounce,  wide-mouthed  stoppered  bottles,  which 
are  to  be  sterilised  by  dry  heat  (the  stopper  must  be  sterilised  separately 
from  the  bottle  and  not  inserted  in  the  latter  till  both  are  cold,  otherwise 
it  will  be  so  tightly  held  as  to  make  removal  very  difficult).  In  using 
such  a  bottle  it  is  best  to  immerse  it  in  the  water  and  then  remove  the 


136  .       BACTERIA  IN   WATER 

stopper  with  forceps.  Care  must  be  taken  not  to  touch  the  water-bed, 
as  the  vegetable  matter  covering  it  contains  a  large  number  of  organisms. 
The  bottles  ought  to  be  packed  in  ice  and  sawdust,  and  plates  must  be 
prepared  from  the  samples  as  soon  as  possible.  When  the  object  in 
view  is  to  determine  the  number  of  bacteria  per  cubic  centimetre,  it  is 
important  to  note  that  water  bacteria  grow  at  very  varied  rates,  and 
therefore  it  is  well  that  the  same  time  should  always  elapse  before  the 
colonies  are  counted.  The  period  of  growing  usually  allowed  is  forty- 
eight  hours  at  20°  C. 

Several  points  may  be  here  noted.  It  has  been  found,  for  instance, 
that  slight  variations  in  the  reaction  of  the  medium  affect  the  number 
of  colonies  which  develop.  A  slightly  greater  degree  of  alkalinity  than 
peptone  gelatin,  as  ordinarily  prepared,  possesses — such  an  increased 
degree  as  that  caused  by  the  addition  of  '01  grm.  Na2C03  to  10  c.c. 
peptone  gelatin — will  give  a  greater  yield  of  colonies  than  the  ordinary 
gelatin.  Again,  the  natural  temperature  of  the  growth  of  water  bacteria 
in  temperate  climates  is  comparatively  low,  being  not  often  above  18°  C., 
and,  on  account  of  this,  gelatin  suggests  itself  as  the  most  suitable 
medium.  This  can  be  seen  by  comparing  the  growth  on  an  agar  plate 
inoculated  with  a  given  quantity  of  water,  and  incubated  at  37°  C.,  with 
the  growth  on  a  precisely  similar  gelatin  plate  incubated  at  20°  C. ,  as  it 
will  be  found  that  many  more  colonies  have  developed  on  the  latter. 
This  fact  may  be  taken  advantage  of  when  pathogenic  bacteria  are  being 
sought  for  in  a  water.  The  latter  usually  grow  well  at  37°  C.,  and  thus 
if  agar  plates  be  used  the  search  may  be  facilitated.  Apart  from  the 
difference  of  incubation  temperatures,  however,  in  such  a  case  as  that 
cited,  it  is  probable  that  agar  is  a  less  suitable  medium  than  gelatin  for 
the  growth  of  water  bacteria,  for  in  plates  incubated  at  the  same 
temperature  the  colonies  which  grow  on  the  agar  are  often  fewer  than 
those  on  the  gelatin.  Probably  no  one  medium  will  support  the  growth 
of  all  the  organisms  present  in  a  given  sample  of  water,  and  under 
certain  circumstances  special  media  must  therefore  be  used.  Thus 
Hansen  found  that  in  testing  waters  to  be  used  in  brewing  it  was 
advisable  to  have  in  the  medium  employed  some  sterile  wort  or  beer,  so 
that  the  organisms  in  the  test  experiments  should  be  provided  with  the 
food  materials  which  would  be  present  in  the  commercial  use  of  the  water. 
Manifestly  this  principle  applies  generally  in  the  bacteriological  examina- 
tion of  waters  to  be  used  for  industrial  purposes. 

In  ordinary  public  health  work  it  may  be  taken  that  the  most 
frequent  and  important  inquiry  is  directed  towards  the  presence  or 
absence  of  the  b.  coli  and  its  congeners.  Many  methods  are  here  used 
but  we  consider  that  in  which  MacConkey's  bile-salt  media  are  employed 
the  most  convenient.  For  small  quantities  of  water, — up  to  1  c.c., — the 
sample  is  simply  added  to  a  Durham's  tube  of  bile-salt  glucose  neutral- 
red  broth  and  incubated  for  48  hours.  When  it  is  necessary  to  examine 
larger  samples  it  is  convenient,  as  Savage  recommends,  to  have  the  bile- 
salt  broth  made  of  double,  treble,  or  quadruple  its  usual  strength. 
The  water  to  be  examined  is  used  as  the  diluent  by  which  the  medium 
is  brought  down  to  the  ordinary  concentration.  If  gas  forms,  some  of 
the  coli  group  are  almost  certainly  present.  The  organisms  may  be 
plated  out  by  smearing  a  little  of  the  broth  on  bile-salt  agar  for  further 
isolation  and  examination. 

With  regard  to  the  objects  with  which   the    bacteriological 


BACTERIA   IN   WATER  137 

examination  of  water  may  be  undertaken,  though  these  may 
be  of  a  purely  scientific  character,  they  usually  aim  at  contribut- 
ing to  the  settlement  of  questions  relating  to  the  potability  of 
waters,  to  their  use  in  commerce,  and  to  the  efficiency  of  pro- 
cesses undertaken  for  the  purification  of  waters  which  have 
undergone  pollution.  The  last  of  these  objects  is  often  closely 
associated  with  the  first  two,  as  the  question  so  often  arises 
whether  a  purification  process  is  so  efficient  as  to  make  the 
water  again  fit  for  use. 

Water  derived  from  any  natural  source  contains  bacteria, 
though,  as  in  the  case  of  some  artesian  wells  and  some  springs, 
the  numbers  may  be  very  small,  e.g.  4  to  100  per  c.c.  In  rain, 
snow,  and  ice  there  are  often  great  numbers,  those  in  the  first  two 
being  derived  from  the  air.  Great  attention  has  been  paid  to 
the  bacterial  content  of  wells  and  rivers.  With  regard  to  the 
former,  precautions  are  necessary  in  arriving  at  a  judgment. 
If  the  water  in  a  well  has  been  standing  for  some  time, 
multiplication  of  bacteria  may  give  a  high  value.  To  meet  this 
difficulty,  if  practicable,  the  well  ought  to  be  pumped  dry  and 
then  allowed  to  fill,  in  order  to  get  at  what  is  really  the  im- 
portant point,  namely,  the  bacterial  content  of  the  water  entering 
the  well.  Again,  if  the  sediment  of  the  well  has  been  stirred 
up  a  high  value  is  obtained.  Ordinary  wells  of  medium  depth 
contain  from  100  to  2000  per  c.c.  With  regard  to  rivers  very 
varied  results  are  obtained.  Moorland  streams  are  usually  very 
pure.  In  an  ordinary  river  the  numbers  present  vary  at 
different  seasons  of  the  year,  whilst  the  prevailing  temperature, 
the  presence  or  absence  of  decaying  vegetation,  or  of  washings 
from  land,  and  dilution  with  large  quantities  of  pure  spring 
water,  are  other  important  features.  Thus  the  Franklands 
found  the  rivers  Thames  and  Lea  purest  in  summer,  and  this 
they  attributed  to  the  fact  that  in  this  season  there  is  most 
spring  water  entering,  and  very  little  water  as  washings  off  land. 
In  the  case  of  other  rivers  the  bacteria  have  been  found  to  be 
fewest  in  winter.  A  great  many  circumstances  must  therefore 
be  taken  into  account  in  dealing  with  mere  enumerations  of 
water  bacteria,  and  such  enumerations  are  only  useful  when 
they  are  taken  stimultaneously  over  a  stretch  of  river,  with 
special  reference  to  the  sources  of  the  water  entering  the  river. 
Thus  it  is  usually  found  that  immediately  below  a  sewage 
effluent  the  bacterial  content  rises,  though  in  a  comparatively 
short  distance  the  numbers  may  markedly  decrease,  and  it  may 
be  that  the  river  as  far  as  numbers  are  concerned  may  appear 
to  return  to  its  previous  bacterial  content.  The  numbers  of 


138  BACTERIA  IN   WATER 

bacteria  present  in  rivers  vary  so  greatly  that  there  is  little  use 
in  quoting  figures,  most  information  being  obtainable  by 
comparative  enumerations  before  and  after  a  given  event  has 
occurred  to  a  particular  water.  Such  a  method  is  thus  of  great 
use  in  estimating  the  efficacy  of  the  filter  beds  of  a  town  water 
supply.  These  usually  remove  from  95  to  98  per  cent  of  the 
bacteria  present,  and  a  town  supply  as  it  issues  from  the  filter 
beds  should  not  contain  more  than  100  bacteria  per  c.c.  Again, 
is  it  found  that  the  storage  of  water  diminishes  the  number  of 
bacteria  present.  The  highest  counts  of  bacteria  per  c.c.  are 
observed  with  sewage ;  for  example,  in  the  London  sewage  the 
numbers  range  from  six  to  twelve  millions. 

Much  more  important  than  the   mere   enumeration    of   the 
bacteria  present  in  a  water  is  the  question  whether  these  include 
forms  pathogenic  to  man.     The  chief  interest  here,  so  far   as 
Europe  is  concerned,  lies  in  the  fact  that  typhoid  fever  is  so 
frequently  water-borne,  but  cholera  and  certain  other  intestinal 
diseases  have  a  similar  source.     The  search  in  waters  for  the 
organisms  concerned  in  these  diseases  is  a  matter  of  the  greatest 
difficulty,    for   each  belongs  to  a  group  of  organisms  morpho- 
logically similar,  very  widespread  in  nature,  and  many  of  which 
have  little  or  no  pathogenic  action.     The  biological  characters 
of  these  organisms  will  be  given  in  the   chapters    devoted   to 
the  diseases  in  question,  but  here  it  may  be   said   that   from 
the    public    health    standpoint    the    making    of    their    being 
found  a  criterion  for  the  condemning  of  a  water  is  impractic- 
able.    There    is    no    doubt    that    the    typhoid    and     cholera 
bacteria   can   exist    for    some    time    in    water — at   least   this 
has  been  found  to  be  the  case  when    sterile   water   has   been 
inoculated  with  these  bacteria.     But  to  what  extent  the  same  is 
true  when  they  are  placed  in  natural  conditions,  which  involve 
their  living  in  the  presence  of  other  organisms,  is  unknown,  for 
it  may  be  safely  said  that  by  no  known  method  can  the  presence 
of  either  be  demonstrated  in  the  complex  mixtures  which  occur 
in  nature.     With  regard  to  the  typhoid  bacillus,    of   late   the 
tendency  has  been  to  seek  for  the  presence  of  indirect  bacterio- 
logical  evidence    which   might   point   in  the  direction    of   the 
possibility   of   the   presence   of   this   organism.     The   methods 
employed  and  the  lines  along  which  such  investigations    have 
gone  have  already  been   alluded    to    in   connection    with   soil. 
The   whole   question   turns    on   the   possibility   of  recognising 
bacteriologically   the    contamination    of    water    with    sewage. 
Klein  and  Houston  here  insist  on  the  fact  that  in  crude  sewage 
the   b.    coli  or  the  members  of    the  coli  group  are  practically 


BACTERIAL   TREATMENT   OF   SEWAGE        139 

never  fewer  than  100,000  per  c.c.,  and  therefore  if  in  a  water 
this  organism  forms  a  considerable  proportion  of  the  total 
number  of  organisms  present,  then  there  is  grave  reason  for 
suspecting  sewage  pollution.  Houston  holds  that  the  nearer  the 
majority  of  the  coli  organisms  in  a  water  approach  to  the  typical 
reactions  of  coli  the  more  likely  is  sewage  contamination  to  be 
present.  The  reactions  regarded  by  him  as  typical  are,  gas 
production  in  gelatin  shake  culture,  production  of  indol, 
clotting  of  milk,  production  of  fluorescence  in  neutral-red  broth, 
acid  and  gas  production  in  lactose  peptone  solution  (v.  b.  coli). 
The  presence  of  b.  coli  in  100  c.c.  of  deep  well  water  or  in 
10  c.c.  of  river  or  shallow  well  water  is  sufficient  to  condemn 
that  water.  As  the  b.  coli  is  fairly  widespread  in  nature,  Klein 
and  Houston  hold  that  valuable  supporting  evidence  is 
found  in  the  presence  of  the  b.  enteritidis  sporogenes  and  of 
the  streptococci,  both  of  which  are  probably  constant  inhabit- 
ants of  the  human  intestine.  •  The  spores  of  the  former  usually 
number  100  per  c.c.  in  sewage,  and  the  presence  of  the  latter 
can  always  be  recognised  in  '001  grm.  of  human  faeces.  The 
deductions  to  be  drawn  from  the  presence  of  these  in  water 
are  the  same  as  those  to  be  drawn  from  their  presence  in  soil. 
A  further  point  here  is  that  it  is  well,  wherever  practicable,  that 
the  indirect  evidence  as  to  the  potability  of  a  water  which  is 
usually  derived  from  chemical  analysis  should  be  supplemented 
by  a  bacteriological  search  for  the  three  groups  of  organisms 
mentioned.  It  has  been  found  that  in  water  artificially  polluted 
with  sewage  containing  them,  they  can  be  detected  by  bacterio- 
logical methods  in  mixtures  from  ten  to  a  hundred  times  more 
dilute  than  those  in  which  the  pollution  can  be  detected  by 
purely  chemical  methods. 

Bacterial  Treatment  of  Sewage. — Of  late  years  the  opinion 
has  been  growing  that  the  most  appropriate  method  of  dealing 
with  the  disposal  of  sewage  is  to  imitate  as  far  as  possible  the 
processes  which  occur  in  nature  for  the  breaking  up  of  organic 
material.  These  practically  depend  entirely  on  bacterial  action. 
Hence  the  rationale  of  the  most  approved  methods  of  sewage 
disposal  is  to  encourage  the  growth  of  bacteria  which  naturally 
exist  in  sewage,  and  which  are  capable  of  breaking  up  organic 
compounds  and  of  converting  the  nitrogen  into  nitrates  and 
nitrites.  The  technique  by  which  this  is  accomplished  is  very 
varied  and  sometimes  rather  empirical,  but  probably  the  general 
principles  underlying  the  different  methods  are  comparatively 
simple.  It  is  probable  that  for  the  complete  destruction  of  the 
organic  matter  of  sewage  both  aerobic  and  anaerobic  bacteria 


140  BACTERIA   IN   WATER 

are  required,  though  on  this  point  there  may  be  some  difference 
of  opinion.  Certainly  very  fair  results  are  obtained  when 
apparently  the  conditions  chiefly  favour  aerobic  organisms  alone. 
This  is  usually  effected  by  running  the  sewage  on  to  beds  of  sand, 
or  preferably  of  coke,  allowing  it  to  stand  for  some  hours,  slowly 
running  the  effluent  out  through  the  bottom  of  the  bed,  and 
leaving  the  bed  to  rest  for  some  hours  before  recharging.  The 
final  result  is  better  if  the  effluent  be  afterwards  run  over  another 
similar  coke-bed.  According  to  some  authorities  the  sewage,  as 
it  runs  into  the  first  bed,  takes  up  from  the  air  considerable  free 
oxygen,  which,  however,  soon  disappears  during  the  stationary 
period,  so  that  on  leaving  the  first  bed  the  sewage  contains  little 
oxygen.  In  the  latter  part  of  its  stay  it  has  thus  been  submitted 
to  anaerobic  conditions.  Further,  while  by  the  passage  of  the 
effluent  out  of  the  first  bed  oxygen  is  sucked  in,  this  rapidly  dis- 
appears, and  during  the  greater  part  of  the  resting  stage  the 
interstices  of  the  bed  are  filled  with  carbonic  acid  gas,  with 
nitrogen  partly  derived  from  the  air,  partly  from  putrefactive 
processes,  and  thus  in  the  filter  anaerobic  conditions  prevail, 
under  which  the  bacteria  can  act  on  the  deposit  left  on  the  coke. 
On  this  latter  point  there  is  difference  of  opinion,  for,  in  examin- 
ing London  sewage,  Clowes  has  found  oxygen  present  in 
abundance  from  four  to  forty  hours  after  the  sewage  has  been 
run  off.  Sometimes  the  treatment  of  the  sewage  consists  in 
allowing  it  continuously  to  trickle  through  sand  or  gravel  or  coke 
beds.  Probably  the  best  results  in  sewage  treatment  are  obtained 
when  it  is  practicable  to  introduce  a  step  where  there  can  be  no 
doubt  that  the  conditions  are  anaerobic.  This  involves  as  a  pre- 
liminary stage  the  treatment  of  the  sewage  in  what  is  called  a 
septic  tank,  and  the  method  has  been  adopted  at  Exeter,  Sutton, 
and  Yeovil  in  this  country,  and  very  fully  worked  at  in  America 
by  the  State  Board  of  Health  of  Massachusetts.  In  the  explana- 
tion given  of  the  rationale  of  this  process,  sewage  is  looked  on  as 
existing  in  three  stages.  (1)  First  of  all,  fresh  sewage — the  newly 
mixed  and  very  varied  material  as  it  enters  the  main  sewers. 
(2)  Secondly,  stale  sewage — the  ordinary  contents  of  the  main 
sewers.  Here  there  is  abundant  oxygen,  and  as  the  sewage  flows 
along  there  occurs  by  bacterial  action  a  certain  formation  of 
carbon  dioxide  and  ammonia  which  combine  to  form  ammonium 
carbonate.  This  is  the  sewage  as  it  reaches  the  purification  works. 
Here  a  preliminary  mechanical  screening  may  be  adopted,  after 
which  it  is  run  into  an  air-tight  tank — the  septic  tank.  (3) 
It  remains  there  for  from  twenty-four  to  thirty-six  hours,  and 
becomes  a  foul-smelling  fluid — the  septic  sewage.  The  chemical 


ANTISEPTICS  141 

changes  which  take  place  in  the  septic  tank  are  of  a  most  complex 
nature.  The  sewage  entering  it  contains  little  free  oxygen,  and 
therefore  the  bacteria  in  the  tank  are  probably  largely  anaerobic, 
and  the  changes  which  they  originate  consist  of  the  formation  of 
comparatively  simple  compounds  of  hydrogen  with  carbon, 
sulphur,  and  phosphorus.  As  a  result  there  is  a  great  reduction 
in  the  amount  of  organic  nitrogen,  of  albuminoid  ammonia,  and 
of  carbonaceous  matter.  The  latter  fact  is  important,  as  the 
clogging  of  ordinary  filter  beds  is  largely  due  to  the  accumulation 
of  such  material,  and  of  matters  generally  consisting  of  cellulose. 
One  further  important  effect  is  that  the  size  of  the  deposited 
matter  is  decreased,  and  therefore  it  is  more  easily  broken  up 
in  the  next  stage  of  the  process.  This  consists  of  running  the 
effluent  from  the  septic  tank  on  to  filter  beds,  preferably  of  coke, 
where  a  further  purification  process  takes  place.  By  this  method 
there  is  first  an  anaerobic  treatment  succeeded  by  an  aerobic ; 
in  the  latter  the  process  of  nitrification  occurs  by  means  of  the 
special  bacteria  concerned.  The  results  are  of  a  satisfactory  nature, 
there  being  often  a  marked  diminution  in  the  number  of  coli 
organisms  present. 

Often  the  effluent  from  a  sewage  purification  system  contains 
as  many  bacteria  as  the  sewage  entering,  but,  especially  by  means 
of  the  septic  tank  method,  there  is  often  a  marked  diminution. 
It  is  said  by  some  that  pathogenic  bacteria  do  not  live  in  sewage. 
The  typhoid  bacillus  has  been  found  to  die  out  when  placed  in 
sewage,  but  it  certainly  can  live  in  this  fluid  for  a  much  longer 
period  than  that  embraced  by  any  purification  method.  Thus 
the  constant  presence  of  b.  coli,  b.  enteritidis,  and  streptococci 
which  has  been  observed  in  sewage  effluents  must  here  still  be 
looked  on  as  indicating  a  possible  infection  with  the  typhoid 
bacillus,  and  it  is  only  by  great  dilution  and  prolonged  exposure 
to  the  conditions  present  in  running  water  that  such  an  effluent 
can  be  again  a  part  of  a  potable  water. 

ANTISEPTICS. 

The  death  of  bacteria  is  judged  of  by  the  fact  that  when 
they  are  placed  on  a  suitable  food  medium  no  development 
takes  place.  Microscopically  it  would  be  observed  that  division 
no  longer  occurred,  and  that  in  the  case  of  motile  species  move- 
ment would  have  ceased,  but  such  an  observation  has  only 
scientific  interest.  From  the  importance  of  being  able  to  kill 
bacteria  an  enormous  amount  of  work  has  been  done  in  the  way 
of  investigating  the  means  of  doing  so  by  chemical  means, 


142  ANTISEPTICS 

and  the  bodies  having  such  a  capacity  are  called  antiseptics.  It 
is  now  known  that  the  activity  of  these  agents  is  limited  to 
the  killing  of  bacteria  outside  the  animal  body,  but  still  even 
this  is  of  high  importance. 

Methods. — These  vary  very  much.  In  early  inquiries  a  great  point 
was  made  of  the  prevention  of  putrefaction,  and  work  was  done  in  the 
way  of  finding  how  much  of  an  agent  must  be  added  to  a  given  solution 
such  as  beef  extract,  urine,  etc.,  in  order  that  the  bacteria  accidentally 
present  might  not  develop  ;  but  as  bacteria  vary  in  their  powers  of  re- 
sistance, the  method  was  unsatisfactory,  and  now  an  antiseptic  is  usually 
judged  of  by  its  effects  on  pure  cultures  of  definite  pathogenic  microbes, 
and  in  the  case  of  a  sporing  bacterium  the  effect  on  both  the  vegetative 
and  spore  forms  is  investigated.  The  organisms  most  used  are  the 
staphylococcus  pyogenes,  streptococcus  pyogenes,  and  the  organisms  of 
typhoid,  cholera,  diphtheria,  and  anthrax — the  latter  being  most  used 
for  testing  the  action  on  spores.  The  best  method  to  employ  is  to  take 
sloped  agar  cultures  of  the  test  organism,  scrape  off  the  growth,  and  mix 
it  up  with  a  small  amount  of  distilled  water  and  filter  this  emulsion 
through  a  plug  of  sterile  glass  wool  held  in  a  small  sterile  glass  funnel, 
add  a  measured  quantity  of  this  fluid  to  a  given  quantity  of  a  solution 
of  the  antiseptic  in  distilled  water,  then  after  the  lapse  of  the  period  of 
observation  to  remove  one  or  two  loopfuls  of  the  mixture  and  place  them 
in  a  great  excess  of  culture  medium.  Here  it  is  preferable  to  use  fluid 
agar,  which  is  then  plated  and  incubated  ;  such  a  procedure  is  prefer- 
able to  the  use  of  bouillon  tubes,  as  any  colonies  developing  can  easily 
be  recognised  as  belonging  to  the  species  of  bacterium  used.  In  dealing 
with  strong  solutions  of  chemical  agents  it  is  necessary  to  be  sure  that 
the  culture  fluid  is  in  great  excess,  so  that  the  small  amount  of  the 
antiseptic  which  is  transferred  with  the  bacteria  may  be  diluted  far 
beyond  the  strength  at  which  it  still  can  have  any  noxious  influence. 
Sometimes  it  is  possible  at  the  end  of  the  period  of  observation  to 
change  the  antiseptic  into  inert  bodies  by  the  addition  of  some  other 
substance  and  then  test  the  condition  of  the  bacteria,  and  if  the  inert 
substances  are  fluid  there  is  no  objection  to  this  proceeding,  but  if  in 
the  process  a  precipitate  results,  then  it  is  better  not  to  have  recourse 
to  such  a  method,  as  sometimes  the  bacteria  are  carried  down  with  the 
precipitate  and  may  escape  the  culture  test.  The  advisability  of,  when 
possible,  thus  chemically  changing  the  antiseptic  was  first  brought  to 
notice  by  the  criticism  of  Koch's  statements  as  to  the  efficacy  of 
mercuric  chloride  in  killing  the  spores  of  the  b.  anthracis.  The  method 
he  employed  in  his  experiments  was  to  soak  silk  threads  in  an  emulsion 
of  anthrax  spores  and  dry  them.  These  were  then  subjected  to  the 
action  of  the  antiseptic,  well  washed  in  water,  and  laid  on  the  surface  of 
agar.  It  was  found,  however,  that  with  threads  exposed  to  a  far  higher 
concentration  of  the  corrosive  sublimate  than  Koch  had  stated  was 
sufficient  to  prevent  growth,  if  the  salt  were  broken  up  by  the  action  of 
ammonium  sulphide  and  this  washed  off',  growth  of  anthrax  still  occurred 
when  the  threads  were  laid  on  agar.  The  explanation  given  was  that 
the  antiseptic  had  formed  analbuminate  with  the  case  of  each  spore,  and 
that  this  prevented  the  antiseptic  from  acting  upon  the  contained 
protoplasm.  Such  an  occurrence  only  takes  place  with  spores,  and  the 
method  given  above,  in  which  the  small  amount  of  antiseptic  adhering 


THE   ACTION   OF   ANTISEPTICS  143 

to  the  bacteria  is  swamped  in  an  excess  of  culture  fluid,  can  safely  be 
followed,  especially  when  a  series  of  antiseptics  is  being  compared. 

Much  attention  has  been  paid  to  the  standardisation  of  antiseptics, 
and  a  watery  solution  of  carbolic  acid  is  now  generally  taken  as  the 
standard  with  which  other  antiseptics  are  compared.  Rideal  and 
Walker  point  out  that  110  parts  by  weight  of  B.  P.  carbolic  acid  equal 
100  parts  by  weight  of  phenol,  and  they  recommend  the  following  method 
of  standardising.  To  5  c.c.  of  a  particular  dilution  of  the  di&infectant 
add  5  drops  of  a  24-hour-old  bouillon  culture  of  the  organism  (usually 
b.  typhosus)  which  has  been  incubated  at  37°  C.  Shake  the  mixture  and 
make  subcultures  every  2^  minutes  to  15  minutes.  Perform  a  parallel 
series  of  experiments  with  carbolic  acid  and  express  the  comparative 
result  in  multiples  of  the  carbolic  acid  doing  the  same  work. 

The  Action  of  Antiseptics. — In  inquiries  into  the  actions  of 
antiseptics  attention  to  a  great  variety  of  factors  is  necessary, 
especially  when  the  object  is  not  to  compare  different  antiseptics 
with  one  another,  but  when  the  absolute  value  of  any  body  is 
being  investigated.  Thus  the  medium  in  which  the  bacteria  to 
be  killed  are  situated,  is  important ;  the  more  albuminous  the 
surroundings  are,  the  greater  degree  of  concentration  is  required. 
Again,  the  higher  the  temperature  at  which  the  action  is  to  take 
place,  the  more  dilute  may  the  antiseptic  be,  or  the  shorter  the 
exposure  necessary  for  a  given  effect  to  take  place.  The  most 
important  factor,  however,  to  be  considered  is  the  chemical 
nature  of  the  substances  employed.  Though  nearly  every  sub- 
stance which  is  not  a  food  to  the  animal  or  vegetable  body  is 
more  or  less  harmful  to  bacterial  life,  yet  certain  bodies  have 
a  more  marked  action  than  others.  Thus  it  may  be  said  that 
the  most  important  antiseptics  are  the  salts  of  the  heavy  metals, 
certain  acids,  especially  mineral  acids,  certain  oxidising  and  re- 
ducing agents,  a  great  variety  of  substances  belonging  to  the 
aromatic  series,  and  volatile  oils  generally.  In  comparing 
different  bodies  belonging  to  any  one  of  these  groups  the 
chemical  composition  or  constitution  is  very  important,  and  if 
such  comparisons  are  to  be  made,  the  solutions  compared  must 
be  equimolecular ;  in  other  words,  the  action  of  a  molecule  of 
one  body  must  be  compared  with  the  action  of  a  molecule  of 
another  body.  This  can  be  done  by  dissolving  the  molecular 
weight  in  grammes  in  say  a  litre  of  water  (see  p.  33).  When 
this  is  done  important  facts  emerge.  Thus,  generally  speaking, 
the  compounds  of  a  metal  of  high  atomic  weight  are  more 
powerful  antiseptics  than  those  of  one  belonging  to  the  same 
series,  but  of  a  lower  atomic  weight.  Among  organic  bodies 
again  substances  with  high  molecular  weight  are  more  powerful 
than  those  of  low  molecular  weight — thus  butyric  alcohol  is  more 
powerful  than  ethylic  alcohol — and  important  differences  among 


144  ANTISEPTICS 

the  aromatic  bodies  are  associated  with  their  chemical  constitu- 
tion. Thus  among  the  cresols  the  ortho-  and  para-bodies  re- 
semble each  other  in  general  chemical  properties,  and  stand  apart 
from  metacresol ;  they  also  are  similar  in  antiseptic  action,  and 
are  much  stronger  than  the  meta-body.  The  same  may  be 
observed  in  the  other  groups  of  ortho-,  meta-,  and  para-bodies. 
Again,  such  a  property  as  acidity  is  important  in  the  action  of  a 
substance,  and,  generally  speaking,  the  greater  the  avidity  of  an 
acid  to  combine  with  an  alkali,  the  more  powerful  an  antiseptic 
it  is.  With  regard  to  oxidising  agents  and  reducing  agents, 
probably  the  possession  of  such  properties  has  been  overrated  as 
increasing  bactericidal  potency.  Thus  in  the  case  of  such  re- 
ducers as  sulphurous  acid  and  formic  acid,  the  effect  is  apparently 
chiefly  due  to  the  fact  that  these  substances  are  acids.  Formic 
acid  is  much  more  efficient  than  formate  of  sodium.  In  the  case 
of  permanganate  of  potassium,  which  is  usually  taken  as  the 
type  of  oxidising  agents  in  this  connection,  it  can  be  shown  that 
the  greater  amount  of  the  oxidation  which  takes  place  when  this 
agent  is  brought  into  contact  with  bacteria  occurs  after  the 
organisms  are  killed.  Such  an  observation  is,  however,  not 
conclusive  as  to  the  non-efficiency  of  the  oxidation  process,  for 
the  death  of  the  bacteria  might  be  due  to  the  oxidation  of  a 
very  small  part  of  the  bacterial  protoplasm.  Apart  from  the 
chemical  nature  of  antiseptic  agents,  the  physical  factors  con- 
cerned in  their  solution,  especially  when  they  are  electrolytes, 
probably  play  a  part  in  their  action.  The  part  played  by  such 
factors  is  exemplified  in  the  important  fact  that  a  strong  solution 
acting  for  a  short  time  will  have  the  same  effect  as  a  weaker 
solution  acting  for  a  longer  time.  From  what  has  been  said  it 
will  be  realised  that  the  real  causes  of  a  material  being  an 
antiseptic  are  very  obscure,  and  at  present  we  can  only  have  a 
remote  idea  of  the  factors  at  work. 

The  Actions  of  certain  Antiseptics. — Here  we  can  only 
briefly  indicate  certain  results  obtained  with  the  more  common 
members  of  the  group. 

Chlorine. — All  the  halogens  have  been  found  to  be  powerful 
antiseptics,  but  from  the  cheapness  with  which  it  can  be  produced 
chlorine  has  been  most  used;  not  only  is  it  the  chief  active 
agent  in  the  somewhat  complex  action  of  bleaching  powder,  but 
it  is  also  the  chief  constituent  of  several  proprietary  substances, 
of  which  "  Electrozone  "  is  a  good  example.  This  last  substance 
is  made  from  electrolysing  sea -water,  when  magnesia  and 
chlorine  being  liberated,  magnesium  hypochlorite  and  magnesium 
chloride  are  formed.  In  the  action  of  this  substance  free  hypo- 


ACTIONS   OF   CERTAIN   ANTISEPTICS         145 

chlorous  acid  is  formed,  and  the  effect  produced  is  thus  similar 
to  that  of  bleaching  powder.  Nissen,  investigating  the  action  of 
the  latter,  found  that  1J  per  cent  killed  typhoid  bacilli  in  faeces ; 
and  Rideal  found  that  1  part  to  400-500  disinfected  sewage  in 
fourteen  minutes,  and  Delepine's  results  show  that  1  part  to  50 
(equal  to  '66  per  cent  of  chlorine)  rapidly  kills  the  tubercle 
bacillus,  and  1  part  to  10  (equal  to  3*3  per  cent)  killed  anthrax 
spores.  Klein  found  that  -05  per  cent  of  chlorine  killed  most 
bacterial  spores  in  five  minutes. 

Iodine  Terchloride. — This  is  a  very  unstable  compound  of 
iodine  and  chlorine,  and  though  it  has  been  much  used  as  an 
antiseptic,  seeing  that  the  substance  only  remains  as  IC13  in 
an  atmosphere  of  chlorine  gas,  it  is  open  to  doubt  whether  the 
effects  described  are  not  due  to  a  very  complicated  action  of 
free  hydrochloric  acid,  hydriodic  acid,  of  joxyacids  of  chlorine 
and  iodine  produced  by  its  decomposition,  and  also,  in  certain 
cases,  of  organic  iodine  compounds  formed  from  its  contact  with 
albuminous  material.  It  is  stated  that  the  action  is  very  potent ; 
a  1  per  cent  solution  is  said  instantly  to  kill  even  anthrax  spores, 
but  if  the  spores  be  in  bouillon,  death  occurs  after  from  ten  to 
twelve  minutes.  In  serum  the  necessary  exposure  is  from  thirty 
to  forty  minutes.  A  solution  of  1-1000  will  kill  the  typhoid, 
cholera,  and  diphtheria  organisms  in  five  minutes. 

Nascent  Oxygen. — This  is  chiefly  available  in  two  ways — firstly, 
when  in  the  breaking  up  of  ozone  the  free  third  atom  of  the 
ozone  molecule  is  seeking  to  unite  with  another  similar  atom ; 
secondly,  when  peroxide  of  hydrogen  is  broken  up  into  water 
and  an  oxygen  atom  is  thereby  liberated.  In  commerce  the 
activity  of  "  Sanitas "  compounds  is  due  to  the  formation  of 
ozone  by  the  slow  oxidation  of  the  resin,  camphor,  and  thymol 
they  contain. 

Per  chloride  of.  Mercury. — Of  all  the  salts  of  the  heavy  metals 
this  has  been  most  widely  employed,  and  must  be  regarded  as 
one  of  the  most  powerful  and  useful  of  known  antiseptics.  In 
testing  its  action  on  anthrax  spores  there  is  no  doubt  that  in  the 
earlier  results  its  potency  was  overrated  from  a  neglect  of  the 
fact  already  alluded  to,  that  in  the  spore-case  an  albuminate  of 
mercury  was  formed  which  prevented  the  contained  protoplasm 
from  developing,  while  not  depriving  it  of  life.  It  has  been 
found,  however,  that  this  salt  in  a  strength  of  1-100  will  kill  the 
spores  in  twenty  minutes,  although  an  hour's  exposure  to  1-1000 
has  no  effect.  The  best  results  are  obtained  by  the  addition  to 
the  corrosive  sublimate  solution  of  -5  per  cent  of  sulphuric  acid 
or  hydrochloric  acid  ;  the  spores  will  then  be  killed  by  a  seventy- 
10 


146  ANTISEPTICS 

minute  exposure  to  a  1-200  solution.  When,  however,  organisms 
in  the  vegetative  condition  are  being  dealt  with,  much  weaker 
solutions  are  sufficient;  thus  anthrax  bacilli  in  blood  will  be 
killed  in  a  few  minutes  by  1-2000,  in  bouillon  by  1-40,000,  and 
in  water  by  1-500,000.  Plague  bacilli  are  killed  by  one  to  two 
minutes'  exposure  to  1-3000.  Generally  speaking,  it  may  be  said 
that  a  1-2000  solution  must  be  used  for  the  practically  instan- 
taneous killing  of  vegetative  organisms. 

Perchloride  of  mercury  is  one  of  the  substances  which  have 
been  used  for  disinfecting  rooms  by  distributing  it  from  a  spray 
producer,  of  which  the  Equifex  may  be  taken  as  a  type.  With 
such  a  machine  it  is  calculated  that  1  oz.  of  perchloride  of 
mercury  used  in  a  solution  of  1-1000  will  probably  disinfect  3000 
square  feet  of  surface.  Such  a  procedure  has  been  extensively 
used  in  the  disinfection  of  plague  houses,  but  the  use  of  a  stronger 
solution  (1-500  acidulated)  is  probably  preferable. 

Formalin  as  a  commercial  article  is  a  40  per  cent  solution  of 
formaldehyde  in  water.  This  is  a  substance  which  of  late  years 
has  come  much  into  vogue,  and  it  is  undoubtedly  a  valuable 
antiseptic.  A  disadvantage,  however,  to  its  use  is  that,  when 
diluted  and  exposed  to  air,  amongst  other  changes  which  it 
undergoes  it  may  be  transformed,  under  little  understood 
conditions,  into  trioxymethylene  and  paraform aldehyde,  these 
being  polymers  of  formaldehyde.  The  bactericidal  values  of  these 
mixtures  are  thus  indefinite.  Formalin  may  be  used  either  by 
applying  it  in  its  liquid  form  or  as  a  spray,  or  the  gas  which 
evaporates  at  ordinary  temperatures  from  the  solution  may  be 
utilised.  To  disinfect  such  an  organic  mixture  as  pus  containing 
pyogenic  organisms  a  10  per  cent  solution  acting  for  half  an 
hour  is  necessary.  In  the  case  of  pure  cultures,  a  5  per  cent 
solution  will  kill  the  cholera  organism  in  three  minutes,  anthrax 
bacilli  in  a  quarter  of  an  hour,  and  the  spores  in  five  hours. 
When  such  organisms  as  pyogenic  cocci,  cholera  spirillum,  and 
anthrax  bacillus  infect  clothing,  an  exposure  to  the  full  strength 
of  formalin  for  two  hours  is  necessary,  and  in  the  case  of  anthrax 
spores,  for  twenty-four  hours.  Silk  threads  impregnated  with 
the  plague  bacillus  were  found  to  be  sterile  after  two  minutes' 
exposure  to  formalin. 

The  action  of  formalin  vapour  has  been  much  studied,  as  its 
use  constitutes  a  cheap  method  of  treating  infected  rooms,  in 
which  case  some  spray-producing  machine  is  employed.  It  is 
stated  that  a  mixture  of  8  c.c.  of  formalin  with  48  c.c.  of  water 
is  sufficient  when  vapourised  to  disinfect  one  cubic  metre,  so  far 
as  non-sporing  organisms  are  concerned.  It  is  stated  that  1  part 


ACTIONS   OF   CERTAIN   ANTISEPTICS          147 

formalin  in  10,000  of  air  will  kill  the  cholera  vibrio  in  one  hour, 
diphtheria  bacillus  in  three  hours,  the  staphylococcus  pyogenes 
in  six  hours,  and  anthrax  spores  in  thirteen  hours.  In  the  case 
of  organisms  which  have  become  dry  it  is  probable,  however, 
that  much  longer  exposures  are  necessary,  but  on  this  point  we 
have  not  definite  information. 

Formalin  gas  has  only  a  limited  application ;  it  has  little 
effect  on  dry  organisms,  and  in  the  case  of  wet  organisms,  in 
order  to  be  effective,  probably  must  become  dissolved  so  as  to 
give  the  moisture  a  proportion  analogous  to  the  strengths  stated 
above  with  regard  to  the  vapour. 

Sulphurous  Acid. — This  substance  has  long  been  in  use, 
largely  from  the  cheapness  with  which  it  can  be  produced  by 
burning  sulphur  in  the  air.  An  atmosphere  containing  *98  per 
cent  will  kill  the  pyogenic  cocci  in  two  minutes  if  they  are  wet, 
and  in  twenty  minutes  if  they  are  dry ;  and  anthrax  bacilli  are 
killed  by  thirty  minutes'  exposure,  but  to  kill  anthrax  spores  an 
exposure  of  from  one  to  two  hours  to  an  atmosphere  containing 
1 1  per  cent  is  necessary.  For  a  small  room  the  burning  of  about 
a  pound  and  a  half  (most  easily  accomplished  by  moistening  the 
sulphur  with  methylated  spirit)  is  usually  considered  sufficient. 
It  has  been  found  that  if  bacteria  are  protected,  e.g.  when 
they  are  in  the  middle  of  small  bundles  of  clothes,  no  effect  is 
produced  even  by  an  atmosphere  containing  a  large  proportion 
of  the  sulphurous  acid  gas.  The  practical  applications  of  this 
agent  are  therefore  limited. 

Potassium  Permanganate. — The  action  of  this  agent  very 
much  depends  on  whether  it  can  obtain  free  access  to  the 
bacteria  to  be  killed  or  whether  these  are  present  in  a  solution 
containing  much  organic  matter.  In  the  latter  case  the  oxidation 
of  the  organic  material  throws  so  much  of  the  salt  out  of  action 
that  there  may  be  little  left  to  attack  the  organisms.  Koch 
found  that  to  kill  anthrax  spores  a  5  per  cent  solution  required 
to  act  for  about  a  day ;  for  most  organisms  a  similar  solution 
acting  for  shorter  periods  has  been  found  sufficient,  and  in  the 
case  of  the  pyogenic  cocci  a  1  per  cent  solution  will  kill  in  ten 
minutes.  There  is  little  doubt  that  such  weaker  solutions  are  of 
value  in  disinfecting  the  throat  on  account  of  their  non-irritating 
properties,  and  good  results  in  this  connection  have  been  obtained 
in  cases  of  diphtheria.  A  solution  of  1  in  10,000  has  been 
found  to  kill  plague  bacilli  in  five  minutes. 

Carbolic  Acid. — Of  all  the  aromatic  series  this  is  the  most 
extensively  employed  antiseptic.  All  ordinary  bacteria  in  the 
vegetative  condition,  and  of  these  the  staphylococcus  pyogenes 


148  ANTISEPTICS 

is  the  most  resistant,  are  killed  in  less  than  five  minutes  by  a 
2-3  per  cent  solution  in  water,  so  that  the  5  per  cent  solution 
usually  employed  in  surgery  leaves  a  margin  of  safety.  But  for 
the  killing  of  such  organisms  as  anthrax  spores  a  very  much 
longer  exposure  is  necessary ;  thus  Koch  found  it  necessary  to 
expose  these  spores  for  four  days  to  ensure  disinfection.  The 
risk  of  such  spores  being  present  in  ordinary  surgical  procedure 
may  be  overlooked,  but  there  might  be  risk  of  tetanus  spores 
not  being  killed,  as  these  will  withstand  fifteen  hours'  exposure 
to  a  5  per  cent  solution. 

In  the  products  of  the  distillation  of  coal  there  occur,  besides 
carbolic  acid,  many  bodies  of  a  similar  chemical  constitution,  and 
many  mixtures  of  these  are  in  the  market — the  chief  being  creolin, 
izal,  and  lysol,  all  of  which  are  agents  of  value.  Of  these  lysol 
is  perhaps  the  most  noticeable,  as  from  its  nature  it  acts  as  a  soap 
and  thus  can  remove  fat  and  dirt  from  the  hands.  A  one-third 
per  cent  solution  is  said  to  destroy  the  typhoid  and  cholera 
organisms  in  twenty  minutes.  A  1  per  cent  solution  is  sufficient 
for  ordinary  surgical  procedures. 

lodoform. — This  is  an  agent  regarding  the  efficacy  of  which 
there  has  been  much  dispute.  There  is  little  doubt  that  it  owes 
its  efficiency  to  its  capacity  for  being  broken  up  by  bacterial 
action  in  such  a  way  as  to  set  free  iodine,  which  acts  as  a  powerful 
disinfectant.  The  substance  is  therefore  of  value  in  the  treatment 
of  foul  wounds,  such  as  those  of  the  mouth  and  rectum,  where 
reducing  bacteria  are  abundantly  present.  It  acts  more  slightly 
where  there  are  only  pyogenic  cocci,  and  it  seems  to  have  a 
specially  beneficial  effect  in  tubercular  affections.  In  certain 
cases  its  action  may  apparently  be  aided  by  the  presence  of  the 
products  of  tissue  degeneration. 

From  the  results  which  have  been  given  it  will  easily  be 
recognised  that  the  choice  of  an  antiseptic  and  the  precise 
manner  in  which  it  is  to  be  employed  depend  entirely  on  the 
environment  of  the  bacteria  which  are  to  be  killed.  In  many 
cases  it  will  be  quite  impossible,  without  original  inquiry,  to  say 
what  course  is  likely  to  be  attended  with  most  success. 


CHAPTEE  V. 

RELATIONS   OF   BACTERIA   TO   DISEASE— THE 
PRODUCTION   OF   TOXINS   BY   BACTERIA. 

Introductory. — It  has  already  been  stated  that  a  strict  division 
of  micro-organisms  into  saprophytes  and  true  parasites  cannot  be 
made.  No  doubt  there  are  organisms  such  as  the  bacillus  of 
leprosy  which  as  yet  have  not  been  cultivated  outside  the  animal 
body,  and  others,  such  as  the  gonococcus,  which  are  in  natural 
conditions  always  parasites  associated  with  disease.  But  these 
latter  can  lead  a  saprophytic  existence  in  specially  prepared 
conditions,  and  there  are  many  of  the  disease-producing  organisms, 
such  as  the  organisms  of  typhoid  and  cholera,  which  can  flourish 
readily  outside  the  body,  even  in  ordinary  conditions.  The 
conditions  of  growth  are,  however,  of  very  great  importance  in 
the  study  of  the  modes  of  infection  in  the  various  diseases, 
though  they  do  not  form  the  basis  of  a  scientific  division. 

A  similar  statement  applies  to  the  terms  pathogenic  and 
saprophytic,  and  even  to  the  terms  pathogenic  and  non-pathogenic. 
By  the  term  pathogenic  is  meant  the  power  which  an  organism 
has  of  producing  morbid  changes  or  effects  in  the  animal 
body,  either  under  natural  conditions  or  in  conditions  artificially 
arranged  as  in  direct  experiment.  Now  we  know  of  no  organ- 
isms which  will  in  all  circumstances  produce  disease  in  all 
animals,  and,  on  the  other  hand,  many  bacteria  described  as 
harmless  saprophytes  will  produce  pathological  changes  if  intro- 
duced in  sufficient  quantity.  When,  therefore,  we  speak  of  a 
pathogenic  organism,  the  term  is  merely  a  relative  one,  and 
indicates  that  in  certain  circumstances  the  organism  will  produce 
disease,  though  in  the  science  of  human  pathology  it  is  often 
used  for  convenience  as  implying  that  the  organism  produces 
disease  in  man  in  natural  conditions. 

Modifying  Conditions. — In  studying  the  pathogenic  effects  in 
149 


150      RELATIONS   OF   BACTERIA   TO   DISEASE 

any  instance,  both  the  micro-organisms  and  the  animal  affected 
must  be  considered,  and  not  only  the  species  of  each,  but  also 
its  exact  condition  at  the  time  of  infection.  In  other  words, 
the  resulting  disease  is  the  product  of  the  sum  total  of  the 
characters  of  the  infecting  agent,  on  the  one  hand,  and  of  the 
subject  of  infection,  on  the  other.  We  may,  therefore,  state  some 
of  the  chief  circumstances  which  modify  each  of  these  two  factors 
involved  and,  consequently,  the  diseased  condition  produced. 

1.  The  Infecting  Agent. — In  the  case  of  a  particular  species 
of  bacterium  its  effect  will  depend  chiefly  upon  (a)  its  virulence, 
and  (6)  the  number  introduced  into  the  body.  To  these  may 
be  added  (c)  the  path  of  infection. 

The  virulence,  i.e.  the  power  of  multiplying  in  the  body  and 
producing  disease,  varies  greatly  in  different  conditions,  and  the 
methods  by  which  it  can  be  diminished  or  increased  will  be 
afterwards  described  (vide  Chapter  XIX.).  One  important 
point  is  that  when  a  bacterium  has  been  enabled  to  invade 
and  multiply  in  the  tissues  of  an  animal,  its  virulence  for  that 
species  is  often  increased.  This  is  well  seen  in  the  case  of 
certain  bacteria  which  are  normally  present  on  the  skin  or 
mucous  surfaces.  Thus  it  has  been  repeatedly  proved  that  the 
bacillus  coli  cultivated  from  a  septic  peritonitis  is  much  more 
virulent  than  that  taken  from  the  bowel  of  the  same  animal. 
The  virulence  may  be  still  more  increased  by  inoculating  from 
one  animal  to  another  in  series — the  method  of  passage.  Widely 
different  effects  are,  of  course,  produced  on  the  virulence  being 
altered.  For  example,  a  streptococcus  which  produces  merely 
a  local  inflammation  or  suppuration,  may  produce  a  rapidly  fatal 
septicaemia  when  its  virulence  is  raised.  Virulence  also  has  a 
relation  to  the  animal  employed,  as  occasionally  on  being  in- 
creased for  one  species  of  animal  it  is  diminished  for  another. 
For  example,  streptococci,  on  being  inoculated  in  series  through 
a  number  of  mice,  acquire  increased  virulence  for  these  animals, 
but  become  less  virulent  for  rabbits.  (Knorr.)  The  theoretical 
consideration  of  virulence  must  be  reserved  for  a  later 
chapter  (see  Immunity). 

The  number  of  the  organisms  introduced,  i.e.  the  dose  of  the 
infecting  agent,  is  another  point  of  importance.  The  healthy 
tissues  can  usually  resist  a  certain  number  of  pathogenic  organisms 
of  given  virulence,  and  it  is  only  in  a  few  instances  that  one  or 
two  organisms  introduced  will  produce  a  fatal  disease,  e.g.  the 
case  of  anthrax  in  white  mice.  The  healthy  peritoneum  of  a 
rabbit  can  resist  and  destroy  a  considerable  number  of  pyogenic 
micrococci  without  any  serious  result,  but  if  a  larger  dose  be 


CONDITIONS   MODIFYING   PATHOGENICITY     151 

introduced,  a  fatal  peritonitis  may  follow.  Again,  a  certain 
quantity  of  a  particular  organism  injected  subcutaneously  may 
produce  only  a  local  inflammatory  change,  but  in  the  case  of  a 
larger  dose  the  organisms  may  gain  entrance  to  the  blood  stream 
and  produce  septicaemia.  There  is,  therefore,  for  a  particular 
animal,  a  minimum  lethal  dose  which  can  be  determined  by 
experiment  only  ;  a  dose,  moreover,  which  is  modified  by  various 
circumstances  difficult  to  control. 

The  path  of  infection  may  alter  the  result,  serious  effects  often 
following  a  direct  entrance  into  the  blood  stream.  Staphylococci 
injected  subcutaneously  in  a  rabbit  may  produce  only  a  local 
abscess,  whilst  on  intravenous  injection  multiple  abscesses  in 
certain  organs  may  result  and  death  may  follow.  Local  inflam- 
matory reaction  with  subsequent  destruction  of  the  organisms 
may  be  restricted  to  the  site  of  infection  or  may  occur  also  in 
the  lymphatic  glands  in  relation.  The  latter  therefore  act  as  a 
second  barrier  of  defence,  or  as  a  filtering  mechanism  which  aids 
in  protecting  against  blood  infection.  This  is  well  illustrated  in 
the  case  of  "  poisoned  wounds."  In  some  other  cases,  however, 
the  organisms  are  very  rapidly  destroyed  in  the  blood  stream, 
and  Klemperer  has  found  that  in  the  dog,  subcutaneous  injection 
of  the  pneumococcus  produces  death  more  readily  than  intra- 
venous injection. 

2.  The  Subject  of  Infection. — Amongst  healthy  individuals 
susceptibility  and,  in  inverse  ratio,  resistance  to  a  particular 
microbe  may  vary  according  to  (a)  species,  (6)  race  and  individual 
peculiarities,  (c)  age.  Different  species  of  the  lower  animals 
show  the  widest  variation  in  this  respect,  some  being  extremely 
susceptible,  others  highly  resistant.  Then  there  are  diseases, 
such  as  leprosy,  gonorrhoea,  etc.,  which  appear  to  be  peculiar  to 
the  human  subject  and  have  not  yet  been  transmitted  to  animals. 
And  further,  there  are  others,  such  as  cholera  and  typhoid,  which 
do  not  naturally  affect  animals,  and  the  typical  lesions  of  which 
cannot  be  experimentally  reproduced  in  them,  or  appear  only 
imperfectly,  although  pathogenic  effects  follow  inoculation  with 
the  organisms.  In  the  case  of  the  human  subject,  differences  in 
susceptibility  to  a  certain  disease  are  found  amongst  different 
races  and  also  amongst  individuals  of  the  same  race,  as  is  well 
seen  in  the  case  of  tubercle  and  other  diseases.  Age  also  plays 
an  important  part,  young  subjects  being  more  liable  to  certain 
diseases,  e.g.  to  diphtheria.  Further,  -at  different  periods  of  life 
certain  parts  of  the  body  are  more  susceptible,  for  example,  in 
early  life,  the  bones  and  joints  to  tubercular  and  acute  suppura- 
tive  affections. 


152     RELATIONS   OF   BACTERIA  TO   DISEASE 

In  increasing  the  susceptibility  of  a  given  individual,  condi- 
tions of  local  or  general  diminished  vitality  play  the  most 
important  part.  It  has  been  experimentally  proved  that 
conditions  such  as  exposure  to  cold,  fatigue,  starvation,  etc., 
all  diminish  the  natural  resistance  to  bacterial  infection.  Rats 
naturally  immune  can  be  rendered  susceptible  to  glanders  by 
being  fed  with  phloridzin,  which  produces  a  sort  of  diabetes,  a 
large  amount  of  sugar  being  excreted  in  the  urine  (Leo). 
Guinea-pigs  may  resist  subcutaneous  injection  of  a  certain  dose 
of  the  typhoid  bacillus,  but  if  at  the  same  time  a  sterilised 
culture  of  the  bacillus  coli  be  injected  into  the  peritoneum,  they 
quickly  die  of  a  general  infection.  Also  a  local  susceptibility 
may  be  produced  by  injuring  or  diminishing  the  vitality  of  a 
part.  If,  for  example,  previous  to  an  intravenous  injection  of 
staphylococci,  the  aortic  cusps  of  a  rabbit  be  injured,  the 
organisms  may  settle  there  and  set  up  an  ulcerative  endocarditis  ; 
or  if  a  bone  be  injured,  they  may  produce  suppuration  at  the 
part,  whereas  in  ordinary  circumstances  these  lesions  would  not 
take  place.  The  action  of  one  species  of  bacterium  is  also  often 
aided  by  the  simultaneous  presence  of  other  species.  In  this 
case  the  latter  may  act  simply  as  additional  irritants  which 
lessen  the  vitality  of  the  tissues,  but  in  some  cases  their  presence 
also  appears  to  favour  the  development  of  a  higher  degree  of 
virulence  of  the  former. 

These  facts,  established  by  experiment  (and  many  others 
might  be  given),  illustrate  the  important  part  which  local  or 
general  conditions  of  diminished  vitality  may  play  in  the  pro- 
duction of  disease  in  the  human  subject.  This  has  long  been 
known  by  clinical  observation.  In  normal  conditions  the  blood 
and  tissues  of  the  body,  with  the  exception  of  the  skin  and 
certain  of  the  mucous  surfaces,  are  bacterium-free,  and  if  a  few 
organisms  gain  entrance,  they  are  destroyed.  But  if  the  vitality 
becomes  lowered  their  entrance  becomes  easier  and  the  possibility 
of  their  multiplying  and  producing  disease  greatly  increased.  In 
this  way  the  favouring  part  played  by  fatigue,  cold,  etc.,  in  the 
production  of  diseases  of  which  the  direct  cause  is  a  bacterium, 
may  be  understood.  It  is  important  to  keep  in  view  in  this  con- 
nection that  many  of  the  inflammation-producing  and  pyogenic 
organisms  are  normally  present  on  the  skin  and  various  mucous 
surfaces.  The  action  of  a  certain  organism  may  devitalise  the 
tissues  to  such  an  extent  as  to  pave  the  way  for  the  entrance  of 
other  bacteria ;  we  may  mention  the  liability  of  the  occurrence 
of  pneumonia,  erysipelas,  and  various  suppurative  conditions  in 
the  course  of  or  following  infective  fevers.  In  some  cases  the 


MODES   OF  BACTERIAL   ACTION  153 

specific  organism  may  produce  lesions  through  which  the  other 
organisms  gain  entrance,  e.g.  in  typhoid,  diphtheria,  etc.  A 
notable  example  of  diminished  resistance  to  bacterial  infection  is 
seen  in  the  case  of  diabetes ;  tuberculosis  and  infection  with 
pyogenic  organisms  are  prone  to  occur  in  this  disease  and  are  of 
a  severe  character.  It  is  not  uncommon  to  find  in  the  bodies  of 
those  who  have  died  from  chronic  wasting  disease,  collections  of 
micrococci  or  bacilli  in  the  capillaries  of  various  organs,  which 
have  entered  in  the  later  hours  of  life ;  that  is  to  say,  the 
bacterium-free  condition  of  the  blood  has  been  lost  in  the  period 
of  prostration  preceding  death. 

The  methods  by  which  the  natural  resistance  may  be  specific- 
ally increased  belong  to  the  subject  of  immunity,  and  are 
decribed  in  the  chapter  on  that  subject. 

Modes  of  Bacterial  Action. — In  the  production  of  disease 
by  micro-organisms  there  are  two  main  factors  involved,  namely, 
(a)  the  multiplication  of  the  living  organisms  after  they  have- 
entered  the  body,  and  (b)  the  production  by  them  of  poisons 
which  may  act  both  upon  the  tissues  around  and  upon  the  body 
generally.  The  former  corresponds  to  infection,  the  latter  is  of 
the  nature  of  intoxication  or  poisoning.  In  different  diseases  one 
of  these  is  usually  the  more  prominent  feature,  but  both  are 
always  more  or  less  concerned. 

1.  Infection  and  Distribution  of  the  JBacteria  in  the  Body. — 
After  pathogenic  bacteria  have  invaded  the  tissues,  or  in  other 
words  after  infection  by  bacteria  has  taken  place,  their  further 
behaviour  varies  greatly  in  different  cases.  In  certain  cases 
they  may  reach  and  multiply  in  the  blood  stream,  producing  a 
fatal  septicaemia.  In  the  lower  animals  this  multiplication  of  the 
organisms  in  the  blood  throughout  the  body  may  be  very  exten 
sive  (for  example,  the  septicaemia  produced  by  the  pneumococcus 
in  rabbits) ;  but  in  septicaemia  in  man,  it  very  seldom,  if  ever, 
occurs  to  so  great  a  degree,  the  organisms  rarely  remain  in  large 
numbers  in  the  circulating  blood,  and  their  detection  in  it  during 
life  by  microscopic  examination  is  rare,  and  even  culture  methods 
may  give  negative  results  unless  a  large  amount  of  blood  is 
used.  In  such  cases,  however,  the  organisms  may  be  found 
post  mortem  lying  in  large  numbers  within  the  capillaries  of 
various  organs,  e.g.  in  cases  of  septicaemia  produced  by  strepto- 
cocci. In  the  human  subject  more  frequently  one  of  two  things 
happens.  In  the  first  place,  the  organisms  may  remain  local, 
producing  little  reaction  around  them,  as  in  tetanus,  or  a  well- 
marked  lesion,  as  in  diphtheria,  pneumonia,  etc.  Or  in  the 
second  place,  they  may  pass  by  the  lymph  or  blood  stream  to 


154     KELATIONS   OF   BACTERIA  TO   DISEASE 

other  parts  or  organs  in  which  they  settle,  multiply,  and  produce 
lesions,  as  in  tubercle. 

2.  Production  of  Chemical  Poisons. — In  all  these  cases  the 
growth  of  the  organisms  is  accompanied  by  the  formation  of 
chemical  products,  which  act  generally  or  locally  in  varying  degree 
as  toxic  substances.  The  toxic  substances  become  diffused 
throughout  the  system,  and  their  effects  are  manifested  chiefly 
by  symptoms  such  as  the  occurrence  of  fever,  disturbances  of 
the  circulatory,  respiratory,  and  nervous  systems,  etc.  In  some 
cases  corresponding  changes  in  the  tissues  are  found,  for  example, 
the  changes  in  the  nervous  system  in  diphtheria,  to  be  after- 
wards described.  The  general  toxic  effects  may  be  so  slight  as 
to  be  of  no  importance,  as  in  the  case  of  a  local  suppuration, 
or  they  may  be  very  intense  as  in  tetanus,  or  again,  less  severe 
but  producing  cachexia  by  their  long  continuance,  as  in  tuber- 
culosis. 

The  occurrence  of  local  tissue  changes  or  lesions  produced  in 
the  neighbourhood  of  the  bacteria,  as  already  mentioned,  is  one 
of  the  most  striking  results  of  bacterial  action,  but  these  also 
must  be  traced  to  chemical  substances  formed  in  or  around  the 
bacteria,  and  either  directly  or  through  the  medium  of  ferments. 
In  this  case  it  is  more  difficult  to  demonstrate  the  mode  of 
action,  for,  in  the  tissues  the  chemical  products  are  formed  by 
the  bacteria  slowly,  continuously,  and  in  a  certain  degree  of 
concentration,  and  these  conditions  cannot  be  exactly  reproduced 
by  experiment.  It  is  also  to  be  noted  that  more  than  one  poison 
may  be  produced  by  a  given  bacterium,  e.g.  the  tetanus 
bacillus  (p.  380).  Further,  it  is  very  doubtful  whether  all  the 
chemical  substances  formed  by  a  certain  bacillus  growing  in  the 
tissues  are  also  formed  by  it  in  cultures  outside  the  body.  The 
separated  toxin  of  diphtheria,  like  various  vegetable  and  animal 
toxins  (vide  infra},  however,  possesses  a  local  toxic  action  of 
very  intense  character,  evidenced  often  by  extensive  necrotic 
change. 

The  injection  of  large  quantities  of  many  different  pathogenic 
organisms  in  the  dead  conditions  results  in  the  production  of  a 
local  inflammatory  change  which  may  be  followed  by  suppura- 
tion, this  effect  being  possibly  brought  about  by  certain  sub- 
stances in  the  bacterial  protoplasm  common  to  various  species, 
or  at  least  possessing  a  common  physiological  action  (Buchner 
and  others).  When  dead  tubercle  bacilli,  however,  are  intro- 
duced into  the  blood  stream,  nodules  do  result  in  certain  parts 
which  have  a  resemblance  to  ordinary  tubercles.  In  this  case 
the  bodies  of  the  bacilli  evidently  contain  a  highly  resistant  and 


TISSUE   CHANGES   PRODUCED   BY   BACTERIA     155 

slowly  acting  substance  which  gradually  diffuses  around  and 
produces  effects  (vide  Tuberculosis). 

Summary. — We  may  say  then  that  the  action  of  bacteria  as 
disease-producers,  as  in  fact  their  power  to  exist  and  multiply  in 
the  living  body,  depends  upon  the  chemical  products  formed 
directly  or  indirectly  by  them.  This  action  is  shown  by  tissue 
changes  produced  in  the  vicinity  of  the  bacteria  or  throughout 
the  system,  and  by  toxic  symptoms  of  great  variety  of  degree  and 
character. 

We  shall  first  consider  the  effects  of  bacteria  on  the  body 
generally,  and  afterwards  the  nature  of  the  chemical  products. 

EFFECTS  OF  BACTEKIAL  ACTION. 

These  may  be  for  convenience  arranged  in  a  tabular  form  as 
follows  : — 

A.  Tissue  Changes. 

(1)  Local  changes,  i.e.  changes  produced  in  the  neigh- 

bourhood of  the  bacteria. 

Position  (a)  At  primary  lesion. 
(6)  At  secondary  foci. 

Character  (a)  Tissue  reactions  )  Acute  or 

(6)  Degeneration  and  necrosis        j  Chronic. 

(2)  Produced  at  a  distance  from  the  bacteria,  directly  or 

indirectly,  by  the  absorption  of  toxins. 

(a)  In  special  tissues — 

(a)  as  the  result  of  damage,  e.g.  nerve  cells  and 

fibres,  secreting  cells,  vessel  walls,  or 
(P)  changes  of  a  reactive  nature  in  the  blood- 
forming  organs. 

(6)  General  anatomical  changes,  the  effects  of  mal- 
nutrition or  of  increased  waste. 

B.  Changes  in  Metabolism. 

The  occurrence  of  fever,  of  errors  of  assimilation  and 
elimination,  etc. 

A.  Tissue  Changes  produced  by  Bacteria. — The  effects  of 
bacterial  action  are  so  various  as  to  include  almost  all  known 
pathological  changes.  However  varied  in  character,  they  may 
be  classified  under  two  main  headings  : — (a)  those  of  a  degenera- 
tive or  necrotic  nature,  the  direct  result  of  damage,  and  (6)  those 


156      RELATIONS   OF   BACTERIA   TO   DISEASE 

of  reactive  nature,  defensive  or  reparative.  The  former  are  the 
expression  of  the  necessary  vulnerability  of  the  tissues,  the  latter 
of  protective  powers  evolved  for  the  benefit  of  the  organism.  In 
the  means  of  defence  both  leucocytes  and  the  fixed  cells  of  the 
tissues  are  concerned.  Both  show  phagocytic  properties,  i.e. 
have  the  power  of  taking  up  bacteria  into  their  protoplasm.  The 
cells  are  guided  towards  the  focus  of  infection  by  chemiotaxis, 
and  thus  we  find  that  different  bacteria  attract  different  cells. 
The  most  rapid  and  abundant  supply  of  phagocytes  is  seen  in 
the  case  of  suppurative  conditions  where  the  neutrophile  leuco- 
cytes of  the  blood  are  chiefly  concerned.  When  the  local  lesion 
is  of  some  extent  there  is  usually  an  increase  of  these  cells  in 
the  blood — a  neutrophile  leucocytosis.  And  further,  recent 
observations  have  shown  that  associated  with  this  there  is  in  the 
bone-marrow  an  increased  number  of  the  mother-cells  of  these 
leucocytes — the  neutrophile  myelocytes.  The  passage  of  the 
neutrophile  leucocytes  from  the  marrow  into  the  blood,  with  the 
resulting  leucocytosis,  is  also  apparently  due  to  the  absorbed 
bacterial  toxins  acting  chemiotactically  on  the  marrow.  These 
facts  abundantly  show  that  the  means  of  defence  is  not  a  mere 
local  mechanism,  but  that  increased  proliferative  activity  in 
distant  tissues  is  called  into  play.  In  addition  to  direct  phagocyt- 
osis by  these  leucocytes,  there  is  now  abundant  evidence  that 
an  important  function  is  the  production  in  the  body  of  bactericidal 
and  other  antagonistic  substances.  In  other  cases  the  cells 
chiefly  involved  are  the  mononuclear  hyaline  leucocytes,  and 
with  them  the  endothelial  cells,  e.g.  of  serous  membranes,  often 
play  an  important  part  in  the  defence ;  this  is  well  seen  in 
typhoid  fever,  where  the  specific  bacillus  appears  to  have  little 
or  no  action  on  the  neutrophile  leucocytes.  In  other  cases, 
again,  the  reaction  is  chiefly  on  the  part  of  the  connective 
cells,  though  their  proliferation  is  always  associated  with  some 
variety  of  leucocytic  infiltration  and  usually  also  with  the  forma- 
tion of  new  blood  vessels.  Such  a  connective  tissue  reaction 
occurs  especially  in  slow  infections  or  in  the  later  stages  of  an 
acute  infection.  The  tissue  changes  resulting  from  cellular 
activity  in  the  presence  of  bacterial  invasion  are  naturally  very 
varied — examples  of  this  will  be  found  in  subsequent  chapters — 
but  they  may  be  said  to  be  manifestations  of  the  two  funda- 
mental processes  of  (a)  increased  functional  activity — movement, 
phagocytosis,  secretion,  etc. — and  (b)  increased  formative  activity 
— cell  growth  and  division.  The  exudation  from  the  blood 
vessels  has  been  variously  interpreted.  There  is  no  doubt  that 
the  exudate  has  bactericidal  properties  and  also  acts  as  a  diluting 


LOCAL   LESIONS  157 

agent,  but  it  must  still  be  held  as  uncertain  whether  the  process 
of  exudation  ought  to  be  regarded  as  primarily  defensive  or  as 
the  direct  result  of  damage  to  the  endothelium  of  the  vessels. 
It  may  also  be  pointed  out  that  the  various  changes  referred  to 
are  none  of  them  peculiar  to  bacterial  invasion  ;  they  are  examples 
of  the  general  laws  of  tissue  change  under  abnormal  conditions, 
and  they  can  all  be  reproduced  by  chemical  substances  in  solution 
or  in  a  particulate  state.  What  constitutes  their  special  feature 
is  their  progressive  or  spreading  nature,  due  to  the  bacterial 
multiplication. 

(1)  Local  Lesions. — In  some  diseases  the  lesion  has  a  special 
site ;  for  example,  the  lesion  of  typhoid  fever  and,  to  a  less 
extent,  that  of  diphtheria.  In  other  cases  it  depends  entirely 
upon  the  point  of  entrance,  e.g.  malignant  pustule  and  the 
conditions  known  as  wound  infections.  In  others  again,  there  is 
a  special  tendency  for  certain  parts  to  be  affected,  as  the  upper 
parts  of  the  lungs  in  tubercle.  In  some  cases  the  site  has  a 
mechanical  explanation. 

When  organisms  gain  an  entrance  to  the  blood  from  a  primary 
lesion,  the  organs  specially  liable  to  be  affected  vary  greatly  in 
different  diseases.  Pyogenic  cocci  show  a  special  tendency  to 
settle  in  the  capillaries  of  the  kidneys  and  produce  miliary 
abscesses,  whilst  these  lesions  rarely  occur  in  the  spleen.  On 
the  other  hand,  the  nodules  in  disseminated  tubercle  or  glanders 
are  much  more  numerous  in  the  spleen  than  in  the  kidneys, 
which  in  the  latter  disease  are  usually  free  from  them.  The 
important  point  is  that  the  position  of  the  disseminated  lesions 
is  not  to  be  explained  by  a  mechanical  process,  such  as  embolism, 
but  depends  upon  a  special  relation  between  the  organisms  and 
the  tissues,  which  may  be  spoken  of  either  as  a  selective  power 
on  the  part  of  the  organisms  or  a  special  susceptibility  of  tissues, 
possibly  in  part  due  to  their  affording  to  the  organisms  more 
suitable  conditions  of  nutriment.  Even  in  the  case  of  the 
lesions  produced  by  dead  tubercle  bacilli,  a  certain  selective 
character  is  observed. 

Acute  Local  Lesions.  —  The  local  inflammatory  reaction 
presents  different  characters  in  different  conditions.  It  may  be 
accompanied  by  abundant  fibrinous  exudation,  or  by  great 
catarrh  (in  the  case  of  an  epithelial  surface),  or  by  hemorrhage, 
or  by  oedema  ;  it  may  be  localised  or  spreading  in  character ;  it 
may  be  followed  by  suppuration,  and  may  be  accompanied  or 
lead  up  to  necrosis  of  the  tissues  of  the  part,  a  good  example  of 
the  latter  event  being  found  in  a  boil.  Examples  will  be  given 
in  subsequent  chapters.  The  necrotic  or  degenerative  changes 


158      RELATIONS   OF   BACTERIA  TO   DISEASE 

affecting  especially  the  more  highly  developed  elements  of  tissues 
are  chiefly  produced  by  the  direct  action  of  the  bacterial  poisons, 
though  aided  by  the  disturbances  of  nutrition  involved  in  the 
vascular  phenomena.  It  may  here  be  pointed  out  that  a  well- 
marked  inflammatory  reaction  is  often  found  •  in  animals  which 
occupy  a  medium  position  in  the  scale  of  susceptibility,  and  that 
an  organism  which  causes  a  general  infection  in  a  certain  animal 
may  produce  only  a  local  inflammation  when  its  virulence  is 
lessened. 

Chronic  Local  Lesions. — In  a  considerable  number  of  diseases 
produced  by  bacteria  the  local  tissue  reaction  is  a  more  chronic 
process  than  those  described.  In  other  words,  the  specific 
irritant  is  less  intense,  so  that  there  is  less  vascular  disturbance 
and  a  greater  preponderance  of  the  proliferative  processes, 
leading  to  new  formation  of  connective  tissue  or  a  modified 
connective  tissue.  This  formation  may  occur  in  foci  here  and 
there,  so  that  nodules  of  greater  or  less  consistence  result,  or  it 
may  be  more  diffuse.  Such  changes  especially  occur  in  the 
diseases  often  known  as  the  infective  granulomata,  of  which 
tubercle,  leprosy,  glanders,  actinomycosis,  syphilis,  etc.,  are 
examples.  A  hard  and  fast  line,  however,  cannot  be  drawn 
between  such  conditions  and  those  described  above  as  acute. 
In  glanders,  for  example,  especially  in  the  human  subject,  the 
lesion  often  approaches  very  nearly  to  an  acute  suppurative 
change,  and  sometimes  actually  is  of  this  nature.  Whilst  in 
these  diseases  the  fundamental  change  is  the  same — viz.  a  re- 
action to  an  irritant  of  minor  intensity — the  exact  structural 
characters  and  arrangement  vary  in  different  diseases.  In  some 
cases  the  disease  may  be  identified  by  the  histological  changes 
alone,  bnt  on  the  other  hand,  this  is  often  impossible.  These 
changes  often  include  the  occurrence  of  degenerations  or  of 
actual  necrosis  in  the  newly  formed  tissue.  In  the  granulomata, 
infection  of  other  parts  from  the  primary  lesion  takes  place 
chiefly  by  the  blood  vessels  and  lymphatics,  though  sometimes 
along  natural  tubes  such  a&  the  bronchi,  intestine,  etc. 

(2)  General  Lesions  produced  by  Toxins. — In  the  various  in- 
fective conditions  produced  by  bacteria,  changes  commonly 
occur  in  certain  organs  unassociated  with  the  presence  of  the 
bacteria;  these  are  produced  by  the  action  of  bacterial  pro- 
ducts circulating  in  the  blood.  Many  such  lesions  can  be  pro- 
duced experimentally.  The  secreting  cells  of  various  organs, 
especially  the  kidney  and  liver,  are  specially  liable  to  change 
of  this  kind.  Cloudy  swelling,  which  may  be  followed  by  fatty 
change  or  by  actual  necrosis  with  granular  disintegration,  is 


DISTURBANCES   OF   METABOLISM,    ETC.       159 

common.  Hyaline  change  in  the  walls  of  arterioles  may  occur, 
and  in  certain  chronic  conditions  waxy  change  is  brought  about 
in  a  similar  manner.  The  latter  has  been  produced  in  animals 
by  the  repeated  injection  of  the  staphylococcus  aureus. 
Capillary  haemorrhages  are  not  uncommon,  and  are  in  many 
cases  due  to  an  increased  permeability  of  the  vessel  walls,  aided 
by  changes  in  the  blood  plasma,  as  evidenced  sometimes  by 
diminished  coagulability.  Similar  haemorrhages  may  follow  the 
injection  of  some  bacterial  toxins,  e.g.  of  diphtheria,  and  also  of 
vegetable  poisons,  e.g.  ricin  and  abrin.  Skin  eruptions  occurring 
in  the  exanthemata  are  probably  produced  in  the  same  way, 
though  in  many  of  these  diseases  the  causal  organism  has  not 
yet  been  isolated.  We  have,  however,  the  important  fact  that 
corresponding  skin  eruptions  may  be  produced  by  poisoning  with 
certain  drugs.  In  the  nervous  system  degenerative  changes 
have  been  found  in  diphtheria,  both  in  the  spinal  cord  and  in 
the  peripheral  nerves,  and  have  been  reproduced  experimentally 
by  the  products  of  the  diphtheria  bacilli.  There  is  also  experi- 
mental evidence  that  the  bacillus  coli  communis  and  the  strepto- 
coccus pyogenes  may,  by  means  of  their  products,  produce  areas 
of  softening  in  the  spinal  cord,  and  this  may  furnish  an  explana- 
tion of  some  of  the  lesions  found  clinically.  It  is  also  possible 
that  some  serous  inflammations  may  be  produced  in  the  same 
way. 

B.  Disturbances  of  Metabolism,  etc. —  It  will  easily  be 
realised  that  such  profound  tissue  changes  as  have  been  detailed 
cannot  occur  without  great  interference  with  the  normal  bodily 
metabolism.  General  malnutrition  and  cachexia  are  of  common 
occurrence,  and  it  is  a  striking  fact  found  by  experiment  that 
after  injection  of  bacterial  products,  e.g.  of  the  diphtheria  bacillus, 
a  marked  loss  of  body  weight  often  occurs  which  may  be  pro- 
gressive, leading  to  the  death  of  the  animal.  In  bacterial 
disease  assimilation  is  often  imperfect,  for  the  digestive  glands 
are  affected,  it  may  be,  by  actual  poisoning  by  bacterial  products, 
it  may  be  by  the  occurrence  of  fever.  The  fatty  degenerations 
which  are  so  common  are  indicative  of  a  breaking  down  of  the 
proteid  molecules,  and  are  associated  with  increased  urea  produc- 
tion, while  the  degeneration  of  the  kidney  epithelium  renders 
the  excretion  of  waste  products  deficient  or  impossible,  and  this 
is  not  infrequently  the  immediate  cause  of  death.  But  of  all 
the  changes  in  metabolism  the  most  difficult  to  understand  is 
the  occurrence  of  that  interference  with  the  heat-regulating 
mechanism  which  results  in  fever.  The  degree  and  course  of  the 
latter  vary,  sometimes  conforming  to  a  more  or  less  definite  type, 


160      RELATIONS   OF   BACTERIA   TO   DISEASE 

where  the  bacilli  are  selective  in  their  field  of  operation,  as  in 
croupous  pneumonia  or  typhoid,  sometimes  being  of  a  very  ir- 
regular kind,  especially  when  the  bacteria  from  time  to  time 
invade  fresh  areas  of  the  body,  as  in  pysemic  affections.  The 
main  point  of  interest  regarding  the  development  of  fever  is  as 
to  whether  it  is  a  direct  effect  of  the  circulation  of  bacterial 
toxins,  or  if  it  is  to  be  looked  on  as  part  of  the  reaction  of  the 
body  against  the  irritant.  This  question  has  still  to  be  settled, 
and  all  that  we  can  do  is  to  adduce  certain  facts  bearing  on  it. 
Thus  in  diphtheria  and  tetanus,  where  toxic  action  leading  to 
degeneration  plays  such  an  important  part,  fever  may  be  a  very 
subsidiary  feature,  except  in  the  terminal  stage  of  the  latter 
disease ;  and  in  fact  in  diphtheria  profoundly  toxic  effects  may 
be  produced  with  little  or  no  interference  with  heat  regulation. 
On  the  other  hand,  in  bacterial  disease,  where  defensive  and  re- 
parative  processes  predominate,  fever  is  rarely  absent,  and  it  is 
nearly  always  present  when  an  active  leucocytosis  is  going  on. 
In  this  connection  it  may  be  remarked  that  several  observers 
have  found  that,  when  a  relatively  small  amount  of  the  dead 
bodies  of  certain  bacteria  are  injected  into  an  animal,  fever 
occurs ;  while  the  injection  of  a  large  amount  of  the  same  is 
followed  by  subnormal  temperatures  and  rapidly  fatal  collapse. 
It  might  appear  as  if  this  indicated  that  the  occurrence  of  fever 
had  a  beneficial  effect,  but  this  is  one  of  the  points  at  issue. 
Certainly  such  an  effect  is  not  due  to  the  bacteria  being  unable 
to  multiply  at  the  higher  degrees  of  temperature  occurring  in 
fever,  for  this  has  been  shown  not  to  be  the  case.  Whether  the 
increase  of  bodily  temperature  indicates  the  occurrence  of 
changes  resulting  in  the  production  of  bactericidal  bodies,  etc., 
is  very  doubtful ;  a  production  of  antagonistic  substances  may 
be  effected  without  the  occurrence  of  fever  or  of  any  appar- 
ent disturbance  of  health.  If  we  consider  the  site  of  the  heat 
production  in  fever  we  again  are  in  difficulties.  It  might  appear 
as  if  the  tissue  destruction,  indicated  by  the  occurrence  of  fatty 
degeneration,  would  lead  to  heat  development,  but  frequently 
excessive  heat  production  with  increased  proteid  metabolism 
occurs  without  any  discoverable  changes  in  the  tissues;  and 
further,  in  phosphorus  poisoning  there  is  little  fever  with  great 
tissue  destruction.  The  increased  work  performed  by  the  heart 
in  most  bacterial  infections  no  doubt  contributes  to  the  rise  of 
bodily  temperature.  But  we  must  bear  in  mind  that  in  fever 
there  is  more  than  mere  increase  of  heat  production — there  is 
also  a  diminished  loss  of  heat  from  interference  with  the  nervous 
mechanism  of  the  sweat  apparatus.  The  known  facts  would 


THE   TOXINS   PRODUCED   BY   BACTERIA      161 

indicate  that  in  fever  there  is  a  factor  involving  the  nervous 
system  to  be  taken  into  account.  The  whole  subject  is  thus 
very  obscure. 

Symptoms. — Many  of  the  symptoms  occurring  in  bacterial 
affections  are  produced  by  the  histological  changes  mentioned, 
as  can  be  readily  understood ;  whilst  in  the  case  of  others,  corre- 
sponding changes  have  not  yet  been  discovered.  Of  the  latter 
those  associated  with  fever,  with  its  disturbances  of  metabolism 
and  manifold  affections  of  the  various  systems,  are  the  most 
important.  The  nervous  system  is  especially  liable  to  be 
affected  —  convulsions,  spasms,  coma,  paralysis,  etc.,  being 
common.  The  symptoms  due  to  disturbance  or  abolition  of  the 
functions  of  secretory  glands  also  constitute  an  important  group, 
forming,  as  they  do,  a  striking  analogy  to  what  is  found  in  the 
action  of  various  drugs. 

These  tissue  changes  and  symptoms  are  given  only  as  illus- 
trative examples,  and  the  list  might  easily  be  greatly  amplified. 
The  important  fact,  however,  is  that  nearly  all,  if  not  quite  all, 
the  changes  found  throughout  the  organs  (without  the  actual 
presence  of  bacteria),  and  also  the  symptoms  occurring  in  infective 
diseases,  can  either  be  experimentally  reproduced  by  the  injection 
of  bacterial  poisons  or  have  an  analogy  in  the  action  of  drugs. 

THE  TOXINS  PRODUCED  BY  BACTERIA. 

Early  Work  on  Toxins. — We  know  that  bacteria  are  capable 
of  giving  rise  to  poisonous  bodies  within  the  animal  body  and 
also  in  artificial  media.  We  know,  however,  comparatively  little 
of  the  actual  nature  of  such  bodies,  and  therefore  we  apply  to 
them  as  a  class  the  general  term  toxins.  The  necessity  for 
accounting  for  the  general  pathogenic  effects  of  certain  bacteria, 
which  in  the  corresponding  diseases  were  not  distributed 
throughout  the  body,  directed  attention  to  the  probable  exist- 
ence of  such  toxins ;  and  the  first  to  systematically  study  the 
production  of  such  poisonous  bodies  was  Brieger.  This  observer 
isolated  from  putrefying  substances,  and  also  from  bacterial 
cultures,  nitrogen-containing  bodies,  which  he  called  ptomaines. 
Similar  bodies  occurring  in  the  ordinary  metabolic  processes  of 
the  body  had  previously  been  described  and  called  leucomaines. 
Ptomaines  isolated  from  pathogenic  bacteria  in  no  case  repro- 
duced the  symptoms  of  the  disease,  except  perhaps  in  tetanus 
and  this  only  owing  to  their  impurity.  The  methods  by 
which  they  were  isolated  were  faulty,  and  they  have  therefore 
only  a  historic  interest. 
11 


162      THE   TOXINS   PRODUCED   BY  BACTERIA 

The  introduction  of  the  principle  of  rendering  fluid  cultures 
bacteria-free  by  nitration  through  unglazed  porcelain,  and  its 
application  by  Roux  and  Yersin  to  obtain,  in  the  case  of  the 
b.  diphtheriae,  a  solution  containing  a  toxin  which  reproduced 
the  symptoms  of  this  disease  (vide  Chap.  XV.),  encouraged  the 
further  inquiry  as  to  the  nature  of  this  toxin.  An  attempt  on 
the  part  of  Brieger  and  Fraenkel  to  obtain  a  purified  diphtheria 
toxin  by  precipitating  bouillon  cultures  by  alcohol  (the  product 
being  denominated  a  toxalbumin)  did  not  greatly  advance  know- 
ledge on  the  subject,  and  further  investigation  soon  showed  that 
characteristic  toxins  can  be  isolated  from  but  few  bacteria. 

General  Facts  regarding  Bacterial  Toxins. — The  following 
may  be  regarded  as  the  chief  facts  regarding  bacterial  toxins 
which  have  been  revealed  by  the  study,  partly  of  the  bodily 
tissues  of  animals  infected  by  the  bacteria  concerned,  partly  of 
artificial  cultures  of  .these  bacteria.  The  dead  bodies  of  certain 
bacteria  have  been  found  to  be  very  toxic.  When,  for  instance, 
tubercle  bacilli  are  killed  by  heat  and  injected  into  the  body 
tissues  of  a  susceptible  animal  tubercular  nodules  are  found 
to  develop  round  the  sites  where  they  have  lodged.  From 
this  it  is  inferred  that  they  must  have  contained  characteristic 
toxins,  seeing  that  characteristic  lesions  result.  The  bodies 
of  the  cholera  vibrio  are  likewise  toxic.  Such  intracellular 
toxins,  as  they  have  been  called,  may  appear  in  the  fluids 
in  which  the  bacteria  are  living  (1)  by  excretion  in  an  un- 
altered or  altered  condition,  (2)  by  the  disintegration  of  the 
bodies  of  the  organisms  which  we  know  are  always  dying 
in  any  bacterial  growth.  The  death  of  bacteria  occurs  also 
in  the  body  of  an  infected  animal,  and  the  disintegration  of 
these  dead  bacteria  constitutes  an  important  means  by  which 
the  poisons  they  contain  are  absorbed.  There  is  some  evidence 
that  often  bacteria  produce  during  growth  poisons  which 
are  hurtful  to  their  own  vitality,  and  also  that  ferments 
are  produced  by  them  which  have  a  solvent  effect  on  the 
poisoned  members  of  the  colony.  Such  a  process  of  autolysis, 
as  it  has  been  called,  may  have  an  important  effect  in 
liberating  intracellular  toxins.  We  do  not,  however,  under- 
stand all  that  takes  place  under  such  circumstances ;  for  the 
dead  bodies  of  many  bacteria,  such  as  those  of  anthrax  and 
diphtheria,  are  relatively  non-toxic.  As  it  is  impossible,  at 
present,  to  obtain  intracellular  toxins  apart  from  other  deriva- 
tives of  the  bacterial  protoplasm,  all  our  knowledge  concerning 
their  effects  is  derived  from  the  study  of  what  happens  when  the 
bodies  of  bacteria  killed  by  chloroform  vapour  or  by  heat  are 


FACTS  REGARDING   BACTERIAL   TOXINS      163 

injected  into  animals.  When  effects  are  produced  by  such 
injections  they  do  not  present  in  any  particular  case  specific 
characters.  They  are  of  the  nature  of  general  disturbances  of 
metabolism,  as  manifested  by  fever,  loss  of  weight,  etc.,  often  of 
such  serious  degree  as  to  result  in  death.  It  is  important  to 
note  that  when  pathogenic  effects  are  produced  these  usually 
appear  very  soon,  it  may  be  in  a  few  hours  after  injection  of  the 
toxic  material ;  there  is  not  the  definite  period  of  incubation 
which  with  other  toxins  often  elapses  before  symptoms  appear. 

Sometimes  the  media  in  which  bacteria  are  growing  become 
extremely  toxic.  This  is  more  marked  in  some  cases  than  in 
others.  The  two  best  examples  of  bacteria  thus  producing 
soluble  toxins  are  the  diphtheria  and  tetanus  bacilli.  In  these 
and  similar  cases  when  bouillon  cultures  are  filtered  bacterium- 
free  by  means  of  a  porcelain  filter,  toxic  fluids  are  obtained, 
which  on  injection  into  animals  reproduce  the  highly  character- 
istic symptoms  of  the  corresponding  diseases.  In  the  case  of 
the  b.  anthracis  and  of  many  others,  at  any  rate  when  grow- 
ing in  artificial  media,  such  toxin  production  is  much  less 
marked,  a  filtered  bouillon  culture  being  relatively  non-toxic. 
Poisons  appearing  in  culture  media  have  been  called  extra- 
cellular toxins,  but  we  cannot  as  yet  say  whether  they  are 
excreted  by  the  bacteria  or  whether  they  are  produced  by 
the  bacteria  acting  on  the  constituents  of  the  media.  We 
therefore  cannot  as  yet  draw  a  hard  and  fast  line  between 
intra-  and  extracellular  toxins,  but  the  terms  are  convenient, 
and  may  apply  to  two  actually  different  sets  of  bodies.  That 
the  poisonous  capacities  of  a  bacterium  may  be  very  compli- 
cated is  shown  by  what  is  known  in  the  case  of  the  cholera 
vibrio,  where  the  poisons  which  dissolve  out  into  the  culture 
fluid  are  probably  different  in  their  nature  from  those  which  act 
when  the  dead  bacteria  are  injected  into  an  animal.  The  extra- 
cellular toxins  are  the  more  easily  obtainable  in  large  quantities, 
and  it  is  their  nature  and  effects  which  are  best  known.  No 
method,  however,  has  been  discovered  of  obtaining  them  in  a 
pure  form,  and  our  knowledge  of  their  properties  is  exclusively 
derived  from  the  study  of  the  toxic  filtrates  of  bouillon  cultures 
— these  filtrates  being  usually  referred  to  simply  as  the  toxins. 
These  toxins  differ  in  their  effects  from  the  intracellular  poisons 
in  that  specific  actions  on  certain  tissues  are  often  manifested. 
Thus  the  toxins  of  the  diphtheria,  the  tetanus,  and  the  botu- 
lismus  bacilli  all  act  on  the  nervous  system ;  with  some  of  the 
pyogenic  bacteria,  on  the  other  hand,  poisons,  probably  of 
similar  nature,  produce  solution  of  red  blood  corpuscles  (this 


164      THE   TOXINS   PRODUCED   BY   BACTERIA 

last  may  explain,  in  part  at  least,  the  anaemias  so  common  in 
the  associated  diseases).  In  the  action  of  many  of  these  toxins 
the  occurrence  of  a  period  of  incubation  between  the  introduction 
of  the  poison  into  the  animal  tissues  and  the  appearance  of 
symptoms  is  often  a  feature. 

We  have  seen  that  in  certain  cases  there  is  difficulty  in  under- 
standing the  action  of  bacteria  which  do  not  form  toxins  in  fluid 
media,  especially  as  in  the  cases  of  some  of  these  the  bacterial 
protoplasm  does  not  seem  very  toxic.  Yet  we  often  see  effects 
produced  at  a  distance  from  the  focus  of  infection,  e.g.  in 
anthrax.  To  explain  such  occurrences  it  has  long  been  put 
forward  as  a  possibility  that  some  bacteria  are  only  capable  of 
producing  toxins  within  the  animal  tissues,  and  it  has  further 
been  thought  possible  that  bacteria,  such  as,  for  example,  the 
typhoid  bacillus,  which  do  in  media  give  rise  to  intracellular 
toxins,  might  either  produce  these  toxins  more  readily  in  the 
tissues  or  might  produce  in  addition  other  toxins  of  a  different 
nature.  Recently  such  toxins  have  been  much  studied,  and  the 
name  aggressins  has  been  given  to  them.  The  evidence  adduced 
for  the  existence  of  these  aggressins  as  a  separate  group  of  bacterial 
poisons  is  of  the  following  kind.  An  animal  is  killed  by  a  dose 
of  the  typhoid,  dysentery,  cholera,  or  tubercle  bacillus,  or  by  a 
staphylococcus,  the  organism  being  introduced  into  one  of  the 
serous  cavities.  After  death  the  serous  exudation,  which  in  all 
these  cases  is  present,  is  removed,  and  centrifugalised  to  remove 
the  bacteria  so  far  as  this  can  be  done  by  such  a  procedure ; 
the  bacteria  which  are  left  are  killed  by  shaking  the  fluid  up  with 
toluol  and  leaving  it  to  stand  for  some  days.  It  is  stated  that 
such  a  fluid  is  of  itself  without  pathogenic  effect,  but  has  the 
property  of  transforming  a  non-lethal  dose  of  the  bacterium  used 
into  one  having  fatal  effect.  Further,  the  effects  of  the  combined 
actions  of  the  bacteria  and  aggressins  are  often  of  a  much  more 
acute  character  than  can  be  obtained  with  toxic  products 
developed  in  vitro.  Thus,  in  the  case  of  the  action  of  a  non- 
lethal  dose  of  the  tubercle  bacillus  plus  its  aggressin,  death  may 
occur  in  twenty  hours,  a  result  never  obtained  with  artificial 
cultures  of  the  organism.  The  results  obtained  are  attributed 
to  a  paralysing  action  which  the  aggressin  is  supposed  to  have 
on  the  phagocytic  functions  of  the  leucocytes.  The  subject  is 
full  of  difficulties,  and  in  the  case  of  certain  of  the  organisms 
employed,  it  is  stated  that  results  similar  to  those  attributed  to 
aggressin  action  have  been  observed  with  macerated  cultures, — 
the  deduction  being  that  in  the  aggressins  we  are  merely  deal- 
ing with  concentrated  intracellular  toxins.  On  the  other  hand, 


THE   NATURE   OF   TOXINS  165 

as  evidence  of  the  existence  of  a  special  group  of  toxins,  it  has 
been  stated  that  a  special  type  of  immunity  against  the 
aggressins  can  be  originated.  Perhaps  the  most  important 
aspect  of  the  controversy  is  the  recognition  of  the  existence  of 
toxins  having  an  action  on  the  leucocytes.  A  poison  causing 
death  of  these  cells  in  connection  with  the  pus-forming  action  of 
the  pyogenic  cocci  has  been  described  under  the  name  of 
leucocidin.  The  investigation  of  such  poisons  must  be  of  the 
highest  importance  in  view  of  the  part  played  by  the  blood- 
cells  in  the  protection  of  the  body  against  infection,  and 
it  is  possible  that  toxins  having  a  fatal  effect  in  strong  con- 
centrations, may,  when  dilute,  be  responsible  for  the  phenomena 
of  attraction  or  repulsion  of  leucocytes  which  we  know  occur 
round  a  focus  of  bacterial  growth  in  the  body. 

It  is  to  be  noted  that  in  the  case  of  any  particular  bacterium 
several  different  toxins  may  be  at  work,  and  it  is  also  possible 
that  one  toxin  may  have  different  effects  on  different  tissues 
of  the  body.  Intracellular  toxins  of  an  organism  may  cause 
general  metabolic  disturbances,  and  its  special  toxins  may  act 
on  special  tissues.  Thus  the  staphylococcus  pyogenes  aureus 
may  cause  fever,  wasting,  etc.,  by  its  intracellular  poisons,  a 
special  action  on  the  leucocytes  by  a  leucocidin  toxin,  and 
anaemia  by  its  hsemolytic  properties.  The  phenomena  of  any 
bacterial  disease  may  thus  in  reality  be  due  to  very  different  and 
complex  causes. 

The  Nature  of  Toxins. — There  is  still  comparatively  little 
known  regarding  this  subject,  and  it  chiefly  relates  to  the  extra- 
cellular toxins.  The  earlier  investigations  upon  toxins  suggested 
that  analogies  exist  between  the  modes  of  bacterial  action  and  what 
takes  place  in  ordinary  gastric' digestion,  and  the  idea  was  worked 
out  for  anthrax,  diphtheria,  tetanus,  and  ulcerative  endocarditis  by 
Sidney  Martin.  This  observer  took,  not  solutions  artificially  made 
up  with  albumoses,1  but  the  natural  fluids  of  the  body  or  definite 

1  In  the  digestion  of  albumins  by  the  gastric  and  pancreatic  juices  the 
albumoses  are  a  group  of  bodies  formed  preliminarily  to  the  production  of 
peptone.  Like  the  latter  they  difl'er  from  the  albumins  in  their  not  being 
coagulated  by  heat,  and  in  being  slightly  dialysable.  They  differ  from  the 
peptones  in  being  precipitated  by  dilute  acetic  acid  in  presence  of  much 
sodium  chloride,  and  also  by  neutral  saturated  sulphate  of  ammonia.  Both 
are  precipitated  by  alcohol.  The  first  albumoses  formed  in  digestion  are 
proto-albumose  and  hetero-albumose,  which  differ  in  the  insolubility  of  the 
latter  in  hot  and  cold  water  (insolubility  and  coagulability  are  quite  different 
properties).  They  have  been  called  the  primary  albumoses.  By  further 
digestion  both  pass  into  the  secondary  albumose,  deutero-albumose,  which 
differs  slightly  in  chemical  reactions  from  the  parent  bodies,  e.g.  it  cannot  be 
precipitated  from  watery  solutions  by  saturated  sodium  chloride  unless  a 


166      THE   TOXINS   PRODUCED   BY   BACTERIA 

solutions  of  albumins,  and,  further,  never  subjected  the  results 
of  the  bacterial  growth  to  heat  above  40°  C.,  or  to  any  stronger 
agent  than  absolute  alcohol.  He  found  that  albumoses  and 
sometimes  peptones  were  formed  by  the  action  of  the  patho- 
genic bacteria  studied,  and  further,  that  the  precipitate  contain- 
ing these  albumoses  was  toxic.  In  certain  cases  the  process  of 
splitting  up  of  the  albumins  went  further  than  in  peptic  diges- 
tion, and  organic  bases  or  acids  might  be  formed.  According  to 
Martin,  the  characteristic  symptoms  of  the  diseases  could  be 
explained  by  compound  actions,  in  which  the  albumoses  were 
responsible  for  some  of  the  effects,  the  remaining  bodies  for 
others.  A  similar  digestive  action  has  been  traced  in  the  case 
of  the  tubercle  bacillus  by  Kuhne. 

Further  evidence  that  bacterial  toxins  are  either  albumoses 
or  bodies  having  a  still  smaller  molecule  is  furnished  by  C.  J. 
Martin.  This  worker,  by  filling  the  pores  of  a  Chamberland 
bougie  with  gelatin,  has  obtained  what  is  practically  a  strongly 
supported  colloid  membrane  through  which  dialysis  can  be  made 
to  take  place  under  great  pressure,  say,  of  compressed  oxygen. 
He  finds  that  in  such  an  apparatus  toxins, — at  least  two  kinds 
tried, — will  pass  through  just  as  an  albumose  will. 

Brieger  and  Boer,  working  with  bouillon  cultures  of  diphtheria 
and  tetanus,  have,  by  precipitation  with  zinc  chloride,  separated 
bodies  which  show  characteristic  toxic  properties,  but  which  have 
the  reactions  neither  of  peptone,  albumose,  nor  album  inate,  and 
the  nature  of  which  is  unknown.  It  has  also  been  found  that 
the  bacteria  of  tubercle,  tetanus,  diphtheria,  and  cholera  can 
produce  toxins  when  growing  in  proteid-free  fluids.  In  the  case 
of  diphtheria  when  the  toxin  is  produced  in  such  a  fluid  a  proteid 
reaction  appears.  Of  course  this  need  not  necessarily  be  caused 
by  the  toxin.  Further  investigation  is  here  required,  for 
Uschinsky,  applying  Brieger  and  Boer's  method  to  a  toxin  so 
produced,  states  that  the  toxic  body  is  not  precipitated  by  zinc 
salts,  but  remains  free  in  the  medium.  If  the  toxins  are  really 
non-proteid  they  may,  on  the  one  hand,  be  the  final  product  of 
a  digestive  action,  or  they  may  be  the  manifestation  of  a  separate 
vital  activity  on  the  part  of  the  bacteria.  On  the  latter  theory 
the  toxicity  of  the  toxic  albumoses  of  Sidney  Martin  may  be  due  to 
the  precipitation  of  the  true  toxins  along  with  these  other  bodies. 
From  the  chemical  standpoint  this  is  quite  possible.  When  we 
take  into  account  the  extraordinary  potency  of  these  poisons  (in 

trace  of  acetic  acid  be  present.  Dysalbnmose  is  probably  merely  a  temporary 
modification  of  hetero-albumose.  Further  digestion  of  deutero- albumose 
results  in  the  formation  of  peptone. 


THE   NATURE   OF   TOXINS  167 

the  case  of  tetanus  the  fatal  dose  of  the  pure  poison  for  a 
guinea-pig  must  often  be  less  than  '000001  gr.),  we  can  under- 
stand how  attempts  by  present  chemical  methods  to  isolate  them 
in  a  pure  condition  are  not  likely  to  be  successful,  and  of  their 
real  nature  we  know  nothing.  In  a  recent  research  Friedberger 
and  Moreschi  have  shown  that  the  intravenous  injection  in  the 
human  subject  of  a  fraction  of  a  loopful  of  a  dead  typhoid 
culture  gives  rise  to  toxic  symptoms,  including  marked  febrile 
reaction.  Such  injections  are  followed  by  the  appearance  of 
agglutinating  and  bacteriolytic  substances  in  the  serum.  These 
results  show  that  intracellular  toxins  may  be  comparable  with 
extra-cellular  toxins  so  far  as  concerns  the  extremely  small  dose 
sufficient  to  produce  toxic  effects. 

Amongst  the  properties  of  the  extracellular  toxins  are 
the  following.  They  are  certainly  all  uncrystallisable ;  they 
are  soluble  in  water  and  they  are  dialysable ;  they  are  pre- 
cipitated along  with  proteids  by  concentrated  alcohol,  and  also 
by  ammonium  sulphate ;  if  they  are  proteids  they  are  either 
albumoses  or  allied  to  the  albumoses ;  they  are  often  relatively  un- 
stable, having  their  toxicity  diminished  or  destroyed  by  heat  (the 
degree  of  heat  which  is  destructive  varies  much  in  different  cases), 
light,  and  by  certain  chemical  agents.  Their  potency  is  often 
altered  in  the  precipitations  practised  to  obtain  them  in  a  pure 
or  concentrated  condition,  but  among  the  precipitants  ammonium 
sulphate  has  little  if  any  harmful  effect.  Regarding  the  toxins 
which  are  more  intimately  associated  with  the  bacterial  proto- 
plasm we  know  much  less,  but  it  is  probable  that  their  nature  is 
similar,  though  some  of  them  at  least  are  not  so  easily  injured  by 
heat,  e.g.  those  of  the  tubercle  bacillus,  already  mentioned.  In 
the  case  of  all  toxins  the  fatal  dose  for  an  animal  varies  with 
the  species,  body  weight,  age,  and  previous  conditions  as  to 
food,  temperature,  etc.  In  estimating  the  minimal  lethal  dose 
of  a  toxin  these  factors  must  be  carefully  considered. 

The  following  is  the  best  method  of  obtaining  concentrated  extra- 
cellular toxins.  The  toxic  fluid  is  placed  in  a  shallow  dish,  and  ammonium 
sulphate  crystals  are  well  stirred  in  till  no  more  dissolve.  Fresh  crystals 
to  form  a  bulk  nearly  equal  to  that  of  the  whole  fluid  are  added,  and  the 
dish  set  in  an  incubator  at  37°  C.  over  night.  Next  day  a  brown  scum 
of  precipitate  will  be  found  floating  on  the  surface.  This  contains  the 
toxin.  It  is  skimmed  off  with  a  spoon,  placed  in  watch  glasses,  and 
these  are  dried  in  vacua  and  stored  in  the  dark,  also  in  vacua,  or  in  an 
exsiccator  containing  strong  sulphuric  acid.  For  use  the  contents  of  one 
are  dissolved  up  in  a  little  normal  saline  solution. 

The  comparison  of  the  action  of  bacteria  in  the  tissues  in 
the  production  of  these  toxins  to  what  takes  place  in  the  gastric 


168      THE   TOXINS   PRODUCED   BY   BACTERIA 

digestion,  has  raised  the  question  of  the  possibility  of  the  elabora- 
tion by  these  bacteria  of  ferments  by  which  the  process  may  be 
started.  Thus  Sidney  Martin  puts  forward  the  view  that 
ferments  may  be  produced,  which  we  may  look  on  as  the 
primary  toxic  agents,  and  which  act  by  digesting  surrounding 
material  and  producing  albumoses, — these  poisons  being,  as  it 
were,  secondary  poisons.  Hitherto  all  attempts  at  the  isolation 
of  bacterial  ferments  of  such  a  nature  have  failed. 

But  apart  from  the  fact  that  with  such  bacteria  as  these  of 
tetanus  and  diphtheria,  a  digestive  action  may  occur,  analogies  have 
been  drawn  between  ferment  and  toxic  action.  The  chief  facts 
upon  which  such  analogies  have  been  founded  are  as  follows. 
Thus  the  toxic  products  of  these  and  other  bacteria  lose  their 
toxicity  by  exposure  to  a  temperature  which  puts  an  end  to  the 
activity  of  such, an  undoubted  ferment  as  that  of  the  gastric 
juice.  If  a  bouillon  containing  diphtheria  toxin  be  heated  at 
65°  C.  for  one  hour,  it  is  found  to  have  lost  much  of  its  toxic 
effect,  and  in  the  case  of  b.  tetani  all  the  toxicity  is  lost  by 
exposure  at  this  temperature.  In  both  diseases  there  is  a  still 
further  fact  which  is  adduced  in  favour  of  the  toxic  substances 
being  of  the  nature  of  ferments,  namely,  the  existence  of  a 
definite  period  of  incubation  between  the  injection  of  the  toxic 
bodies  and  the  appearance  of  symptoms.  This  may  be  inter- 
preted as  showing  that  after  the  introduction  of,  say,  a  filtered 
bouillon  culture,  further  chemical  substances  are  formed  in  the 
body  before  the  actual  toxic  effect  is  produced.  Too  much 
reliance  must  not  be  placed  on  such  an  argument,  for  in  the 
case  of  tetanus,  at  least,  the  delay  may  be  explained  by  the  fact 
that  the  poison  apparently  has  to  travel  up  the  nerve  trunks 
before  the  real  poisonous  action  is  developed.  Further,  with 
some  poisons  presently  to  be  mentioned  which  are  closely  allied 
to  the  bacterial  toxins  an  incubation  period  may  not  exist. 
It  would  not  be  prudent  to  dogmatise  as  to  whether  the  toxins 
do  or  do  not  belong  to  such  an  ill-defined  group  of  substances 
as  the  ferments.  It  may  be  pointed  out,  however,  that  the 
essential  concept  of  a  ferment  is  that  of  a  body  which  can 
originate  change  without  itself  being  changed,  and  no  evidence 
has  been  adduced  that  toxins  fulfil  this  condition.  Another 
property  of  ferments  is  that  so  long  as  the  products  of  fermenta- 
tion are  removed,  the  action  of  a  given  amount  of  ferment  is 
indefinite.  Again,  in  the  case  of  toxins  no  evidence  of  such  an 
occurrence  has  been  found.  A  certain  amount  of  a  toxin  is 
always  associated  with  a  given  amount  of  disease  effect,  though 
a  process  of  elimination  of  waste  products  must  be  all  the  time 


VEGETABLE   AND   ANIMAL   POISONS          169 

going  on  in  the  animal's  body.  Again,  too  much  importance 
must  not  be  attached  to  loss  of  toxicity  by  toxins  at  relatively 
low  temperatures.  This  is  not  true  of  all  toxins,  and  further- 
more many  proteids  show  a  tendency  to  change  at  such 
temperatures ;  for  instance,  if  egg  albumin  be  kept  long 
enough  at  55°  C.  nearly  the  whole  of  it  will  be  coagulated. 
We  must  therefore  maintain  an  open  mind  on  this  subject. 

Similar  Vegetable  and  Animal  Poisons.  —Within  recent  years  it  has 
been  found  that  the  bacterial  poisons  belong  to  a  group  of  toxic  bodies 
all  presenting  very  similar  properties,  other  members  of  which  occur 
widely  in  the  vegetable  and  animal  kingdoms.  Among  plants  the  best- 
known  examples  are  the  ricin  and  abrin  poisons  obtained  by  making 
watery  emulsions  of  the  seeds  of  the  Ricinus  communis  and  the  Abrus 
precatorius  (jequirity)  respectively.  From  the  Robinia  pseudacacia 
another  poison — robin — belonging  to  the  same  group  is  obtained.  The 
chemical  reactions  of  ricin  and  abrin  correspond  to  those  of  the  bacterial 
toxins.  They  are  soluble  in  water,  they  are  precipitable  by  alcohol,  but 
being  less  easily  dialysable  than  the  albumoses  they  have  been  called 
toxalbumins.  Their  toxicity  is  seriously  impaired  by  boiling,  and  they 
also  gradually  become  less  toxic  on  being  kept.  Both  are  among  the 
most  active  poisons  known — ricin  being  the  more  powerful.  When  they 
are  injected  subcutaneously  a  period  of  twenty-four  hours  usually  elapses 
— whatever  be  the  dose — before  symptoms  set  in.  Both  tend  to  produce 
great  inflammation  at  the  seat  of  inoculation,  which  in  the  case  of  ricin 
may  end  in  an  acute  necrosis  ;  in  fatal  cases  hsemorrhagic  enteritis  and 
nephritis  may  be  found.  Both  act  as  irritants  to  mucous  membranes, 
abrin  especially  being  capable  of  setting  up  most  acute  conjunctivitis. 

It  is  also  certain  that  the  poisons  of  scorpions  and  of  poisonous  snakes 
belong  to  the  same  group.  The  poisons  derived  from  the  latter  are 
usually  called  venins,  and  a  very  representative  group  of  such  venins 
derived  from  different  species  has  been  studied.  To  speak  generally 
there  is  derivable  from  the  natural  secretions  of  the  poison  glands  a 
series  of  venins  which  have  all  the  reactions  of  the  bodies  previously 
considered.  Like  ricin  and  abrin,  they  are  not  so  easily  dialysable  as 
bacterial  toxins,  and  therefore  have  also  been  classed  as  toxalbumins. 
Their  properties  are  also  similar  ;  many  of  them  are  destroyed  by  heat, 
but  the  degree  necessary  here  also  varies  much,  and  some  will  stand 
boiling.  There  is  also  evidence  that  in  a  crude  venin  there  may  be  several 
poisons  differently  sensitive  to  heat.  All  the  venins  are  very  powerful 
poisons,  but  here  there  is  practically  no  period  of  incubation — the  effects 
are  almost  immediate.  An  outstanding  feature  of  the  venins  is  the 
complexity  of  the  crude  poison  secreted  by  any  particular  species  of 
snake.  C.  J.  Martin  in  summing  up  the  results  of  many  observers  has 
pointed  out  that  different  venoms  have  been  found  to  contain  one  or 
more  of  the  following  poisons  :  a  neurotoxin  acting  on  the  respiratory 
centre,  a  neurotoxiu  acting  on  the  nerve-endings  in  muscle,  a  toxin 
causing  haemolysis,  toxins  acting  on  other  cells,  e.g.  the  endothelium  of 
blood-vessels  (this  from  its  effects  has  been  named  hsemorrhagin), 
leucocytes,  nerve-cells,  a  toxin  causing  thrombosis,  a  toxin  having  an 
opposite  effect  and  preventing  coagulation,  a  toxin  neutralising  the 
bactericidal  qualities  of  the  body  fluids  and  thus  favouring  putrefaction, 
a  toxin  causing  agglutination  of  the  red  blood  corpuscles,  a  proteolytic 


170      THE   TOXINS   PRODUCED   BY  BACTERIA 

ferment,  a  toxin  causing  systolic  standstill  of  the  excised  heart.  Any 
particular  venom  contains  a  mixture  in  varying  proportions  of  such 
toxins,  and  the  different  effects  produced  by  the  bites  of  different  snakes 
largely  depend  on  this  variability  of  composition.  The  neurotoxic,  the 
thrombotic,  and  the  hsemolytic  toxins  are  very  important  constituents 
of  any  venom.  The  toxicity  of  different  venoms  varies  much,  and  no 
general  statement  can  be  made  with  regard  to  the  toxicity  of  different 
poisons  towards  man.  Lamb  has  calculated  that  the  fatal  dose  of  crude 
cobra  venom  for  man  is  probably  about  -015  of  a  gramme,  and  that  if 
such  a  snake  bites  with  full  glands  many  times  this  dose  would 
probably  be  injected,  but,  of  course,  the  amount  emitted  depends  largely 
on  the  period  which  has  elapsed  since  the  animal  last  emptied  its  glands. 
When  a  dose  of  a  venom  not  sufficient  to  cause  immediate  death  from 
general  effects  be  given,  very  rapid  and  widespread  necrosis  often  may 
occur  in  a  few  hours  round  the  site  of  inoculation. 

An  extremely  important  fact  was  discovered  by  Flexner  and  Noguchi, 
namely,  that  the  hsemolytic  toxin  of  cobra  venom  in  certain  cases  has  no 
action  by  itself,  but  produces  rapid  solution  of  red  corpuscles  when  some 
normal  serum  is  added,  the  latter  containing  a  labile  complement-like 
body,  which  activates  the  venom.  In  this  there  is  a  close  analogy  to 
what  holds  in  the  case  of  a  hsemolytic  serum  deprived  of  complement  by 
heat  at  55°  C.  (p.  479).  Kyes  and  Sachs  further  showed  that  in  addition 
to  serum-complement  a  substance  with  definitely  known  constitution, 
namely  lecithin,  had  the  property  of  activating  the  hsemoly  tic  substance 
in  cobra  venom,  the  two  apparently  uniting  to  form  an  actively  toxic 
substance.  Later  still,  Kyes  succeeded  in  demonstrating  the  union  of 
the  two  substances  to  form  a  cobra-lecithid,  and  in  separating  the 
latter  as  a  practically  pure  compound,  which  is,  unlike  lecithin, 
insoluble  in  ether,  but  soluble  in  chloroform.  So  far  no  example  of 
activating  a  bacterial  toxin  is  known,  but  the  results  mentioned  point  to 
the  possibility  of  this  occurring  in  some  cases  in  the  tissues  of  the  body. 

The  Theory  of  Toxic  Action. — While  we  know  little  of  the 
chemical  nature  of  any  toxins  we  may,  from  our  knowledge  of 
their  properties,  group  together  the  tetanus  and  diphtheria 
poisons,  ricin,  abrin,  snake  poisons,  and  scorpion  poisons. 
Besides  the  points  of  agreement  already  noted,  all  possess  the 
further  property  that,  as  will  be  afterwards  described,  when 
introduced  into  the  bodies  of  susceptible  animals  they  stimulate 
the  production  of  substances  called  antitoxins.  The  nature  of 
the  antagonism  between  toxin  and  antitoxin  will  be  discussed 
later.  Here,  to  explain  what  follows  it  may  be  stated  (1)  that  the 
molecule  of  toxin  most  probably  forms  a  chemical  combination 
with  the  molecule  of  antitoxin,  and  (2)  that  it  has  been  shown 
that  toxin  molecules  may  lose  much  of  their  toxic  power  and 
still  be  capable  of  uniting  with  exactly  the  same  proportion  of 
antitoxin  molecules.  From  these  and  other  circumstances  Ehrlich 
has  advanced  the  view  that  the  toxin  molecule  has  a  very  com- 
plicated structure,  and  contains  two  atom  groups.  One  of  these, 
the  haptophorous  (ctTrretv,  to  bind  to),  is  that  by  which  com- 


THE   THEORY   OF   TOXIC   ACTION  171 

bination  takes  place  with  the  antitoxin  molecule,  and  also  with 
presumably  corresponding  molecules  naturally  existing  in  the 
tissues.  The  other  atom  group  he  calls  the  toxophorous,  and  it 
is  to  this  that  the  toxic  effects  are  due.  This  atom  group  is 
bound  to  the  cell  elements,  e.g.  the  nerve  cells  in  tetanus,  by  the 
haptophorous  group.  Ehrlich  explains  the  loss  of  toxicity  which 
with  time  occurs  in,  say,  diphtheria  toxin,  on  the  theory  that  the 
toxophorous  group  undergoes  disintegration.  And  if  we  suppose 
that  the  haptophorous  group  remains  unaffected  we  can  then 
understand  how  a  toxin  may  have  its  toxicity  diminished  and 
still  require  the  same  proportion  of  antitoxin  molecules  for  its 
neutralisation.  To  the  bodies  whose  toxophorous  atom  groups 
have  become  degenerated,  Ehrlich  gives  the  name  toxoids.  The 
theory  may  afford  an  explanation  of  what  has  been  suspected, 
namely,  that  in  some  instances  toxins  derived  from  different 
sources  may  be  related  to  one  another.  For  example,  Ehrlich 
has  pointed  out  that  ricin  produces  in  a  susceptible  animal  body 
an  antitoxin  which  corresponds  almost  completely  with  that 
produced  by  another  vegetable  poison,  robin  (vide  supra), 
though  ricin  and  robin  are  certainly  different.  This  may  be  ex- 
plained according  to  the  view  that  robin  is  a  toxoid  of  ricin,  i.e. 
their  haptophorous  groups  correspond,  while  their  toxophorous 
differ.  The  evidence  on  which  Ehrlich's  deductions  are  based 
is  of  a  very  weighty  character,  and  will  be  again  referred  to 
in  the  chapter  on  Immunity. 

With  regard  to  the  intracellular  toxins  we  shall  see  it  is 
difficult  to  determine  whether  or  not  they  share  with  the  extra- 
cellular poisons  the  property  of  stimulating  antitoxin  formation, 
— if  they  do  not,  then  they  may  belong  to  an  entirely  different 
class  of  substances.  It  is  certain  that  a  tolerance  against  such 
poisons  is  difficult  to  establish  and  is  not  of  a  lasting  character. 
We  thus  cannot  say  what  the  mechanism  is  by  which  these 
poisons  act.  It  may  be  said  that  Macfadyen  by  grinding  up 
typhoid  bacilli  frozen  by  liquid  air  claimed  that  on  thawing  he 
obtained  the  intracellular  toxins  in  liquid  form,  and  he  further 
stated  that  by  using  this  fluid  he  could  immunise  animals  not 
only  against  the  toxins  but  also  against  the  living  bacteria. 

We  have  already  pointed  out  that  those  who  claim  for  the 
aggressins  a  special  character  hold  that  the  activity  of  these 
bodies  has  as  its  effect  the  interference  with  the  phagocytic 
functions  of  the  leucocytes.  They  also  hold  that  a  special  type 
of  immunity  can  be  developed  against  the  aggressive  bodies. 


CHAPTER   VI. 
INFLAMMATORY   AND   SUPPURATIVE   CONDITIONS. 

THIS  subject  is  an  exceedingly  wide  one,  and  embraces  a  great 
many  pathological  conditions  which  in  their  general  characters 
and  results  are  widely  different.  Thus  in  addition  to  suppuration, 
various  inflammations,  ulcerative  endocarditis,  septicaemia  and 
pyaemia,  will  come  up  for  consideration.  With  regard  to  these 
the  two  following  general  statements,  established  by  bacteriological 
research,  may  be  made  in  introducing  the  subject.  In  the  first 
place,  there  is  no  one  specific  organism  for  any  one  of  these 
conditions;  various  organisms  may  produce  them,  and  not  in- 
frequently more  than  one  organism  may  be  present  together. 
In  the  second  place,  the  same  organism  may  produce  widely 
varying  results  under  different  circumstances, — at  one  time  a 
local  inflammation  or  abscess,  at  another  multiple  suppurations 
or  a  general  septicaemia.  The  principles  on  which  this  diversity 
in  results  depends  have  already  been  explained  (p.  151). 
Furthermore,  there  are  conditions  like  acute  pneumonia,  epidemic 
meningitis,  acute  rheumatism,  etc.,  which  have  practically  the 
character  of  specific  diseases  and  yet  which  as  regards  their 
essential  pathology  belong  to  the  same  class.  The  arrangement 
followed  is  to  a  certain  extent  one  of  convenience. 

It  may  be  well  to  emphasise  some  of  the  chief  points  in  the 
pathology  of  these  conditions.  In  suppuration  the  two  main 
phenomena  are — (a)  a  progressive  immigration  of  leucocytes, 
chiefly  of  the  polymorpho-nuclear  (neutrophile)  variety,  and  (b) 
a  liquefaction  or  digestion  of  the  supporting  elements  of  the 
tissue  along  with  necrosis  of  the  cells  of  the  part.  The  result 
is  that  the  tissue  affected  becomes  replaced  by  the  cream-like 
fluid  called  pus.  A  suppurative  inflammation  is  thus  to  be 
distinguished  on  the  one  hand  from  an  inflammation  without 
destruction  of  tissue,  and  on  the  other  from  necrosis  or  death 

172 


NATURE   OF   SUPPURATION  173 

en  masse,  where  the  tissue  is  not  liquefied,  and  leucocyte 
accumulation  may  be  slight.  When,  however,  suppuration  is 
taking  place  in  a  very  dense  fibrous  tissue,  liquefaction  may  be 
incomplete,  and  a  portion  of  dead  tissue  or  slough  may  remain 
in  the  centre,  as  is  the  case  in  boils.  In  the  case  of  suppuration 
in  a  serous  cavity  the  two  chief  factors  are  the  progressive 
leucocytic  accumulation  and  the  disappearance  of  any  fibrin 
which  may  be  present. 

Many  experiments  have  been  performed  to  determine  whether 
suppuration  can  be  produced  in  the  absence  of  micro-organisms 
by  various  chemical  substances,  such  as  croton  oil,  nitrate  of 
silver,  turpentine,  etc. — care,  of  course,  being  taken  to  ensure 
the  absence  of  bacteria.  The  general  result  obtained  by  in- 
dependent observers  is  that  as  a  rule  suppuration  does  not  follow, 
but  that  in  certain  animals  and  with  certain  substances  it  may, 
the  pus  being  free  from  bacteria.  It  is  still,  however,  questioned 
by  some  whether  the  pus  thus  produced  really  corresponds 
histologically  and  chemically  with  that  due  to  bacterial  action. 
Buchner  showed  that  suppuration  may  be  produced  by  the 
injection  of  dead  bacteria,  e.g.  sterilised  cultures  of  bacillus 
pyocyaneus,  etc.  The  subject  has  now  more  a  scientific  than  a 
practical  interest,  and  the  general  statement  may  be  made  that 
practically  all  cases  of  true  suppuration  met  with  clinically  are 
due  to  the  action  of  living  micro-organisms. 

The  term  septiccemia  is  applied  to  conditions  in  which  the 
organisms  multiply  within  the  blood  and  give  rise  to  symptoms 
of  general  poisoning,  without,  however,  producing  abscesses  in  the 
organs.  In  all  cases  of  septicaemia  the  organisms  are  more 
numerous  in  the  capillaries  of  internal  organs  than  in  the 
peripheral  circulation,  and,  in  the  case  of  the  human  subject,  it 
may  be  impossible  to  detect  any  in  the  blood  during  life,  though 
they  may  be  seen  in  large  numbers  in  the  capillaries  of  the 
kidneys,  liver,  etc.,  post  mortem.  The  essential  fact  m  pyaemia,  on 
the  other  hand,  is  the  occurrence  of  multiple  abscesses  in  internal 
organs  and  other  parts  of  the  body.  In  most  of  the  cases  of 
typical  pyaemia,  common  in  pre-antiseptic  days,  the  starting-point 
of  the  disease  was  a  septic  wound  with  bacterial  invasion  of  a 
vein  leading  to  thrombosis  and  secondary  embolism.  Multiple 
foci  of  suppuration  may  be  produced,  however,  in  other  ways,  as 
will  be  described  below  (p.  186).  If  the  term  "pyaemia"  be 
used  to  embrace  all  such  conditions,  their  method  of  production 
should  always  be  distinguished. 


174         INFLAMMATION   AND   SUPPURATION 


BACTERIA  AS  CAUSES  OF  INFLAMMATION  AND  SUPPURATION. 

A  considerable  number  of  species  of  bacteria  have  been 
found  in  acute  inflammatory  and  suppurative  conditions,  and  of 
these  many  have  been  proved  to  be  causally  related,  whilst  of 
some  others  the  exact  action  has  not  yet  been  fully  determined. 

Ogston,  who  was  one  of  the  first  to  study  this  question  (in 
1881),  found  that  the  organisms  most  frequently  present  were 
micrococci,  of  which  some  were  arranged  irregularly  in  clusters 
(staphylococci),  whilst  others  formed  chains  (streptococci).  He 
found  that  the  former  were  more  common  in  circumscribed  acute 
abscesses,  the  latter  in  spreading  suppurative  conditions.  Rosen- 
bach  shortly  afterwards  (1884),  by  means  of  cultures,  differentiated 
several  varieties  of  micrococci,  to  which  he  gave  the  following 
special  names :  staphylococcus  pyogenes  aureus,  staphylococcus 
pyogenes  albus,  streptococcus  pyogenes,  micrococcus  pyogenes  tennis. 
Other  organisms  are  met  with  in  suppuration,  such  as  staphylo- 
coccus pyogenes  citreus,  staphylococcus  cereus  albus,  staphylococcus 
cereus  flavus,  pneumococcus,  pneumobacillus  (Friedlander),  bacillus 
pyogenes  foetidus  (Passet),  bacillus  coli  communis,  bacillus  lactis 
cerogenes,  bacillus  cerogenes  encapsulatus,  bacillus  pyocyaneus, 
micrococcus  tetragenus,  pneumococcus,  pneumobacillus,  diplococcus 
intracellularis  meningitidis,  and  others. 

In  secondary  inflammations  and  suppurations  following  acute 
diseases  the  corresponding  organisms  have  been  found  in  some 
cases,  such  as  gonococcus,  typhoid  bacillus,  influenza  bacillus,  etc. 
Suppuration  is  also  produced  by  the  actinomyces  and  the 
glanders  bacillus,  and  sometimes  chronic  tubercular  lesions  have 
a  suppurative  character. 

Staphylococcus  Pyogenes  Aureus. — Microscopical  Characters. 
— This  organism  is  a  spherical  coccus  about  '9  /x  in  diameter, 
which  grows  irregularly  in  clusters  or  masses  (Fig.  52).  It  stains 
readily  with  all  the  basic  aniline  dyes,  and  retains  the  colour  in 
Gram's  method. 

Cultivation. — It  grows  readily  in  all  the  ordinary  media  at 
the  room  temperature,  though  much  more  rapidly  at  the 
temperature  of  the  body.  In  stab  cultures  in  peptone  gelatin 
a  streak  of  growth  is  visible  on  the  day  after  inoculation,  and 
on  the  second  or  third  day  liquefaction  commences  at  the  top. 
As  liquefaction  proceeds,  the  growth  falls  to  the  bottom  as  a 
flocculent  deposit,  which  soon  assumes  a  bright  yellow  colour, 
while  a  yellowish  film  may  form  on  the  surface,  the  fluid  portion 
still  remaining  turbid.  Ultimately  liquefaction  extends  out  to 


STAPHYLOCOCCUS  PYOGENES  AUREUS   175 


the  wall  of  the  tube  (Fig.  53).  In  gelatin  plates  colonies  may 
be  seen  with  the  low  power  of  the  microscope  in  twenty-four 
hours,  as  little  balls  somewhat  granular  on  the  surface  and  of 
brownish  colour.  On  the  second  day  they  are  visible  to  the 


- 


FIG.  52. — Staphylococcus  pyogeues  aureus, 
young  culture  on  agar,  showing  clumps 
of  cocci. 

Stained  with  weak  carbol-fuchsin.     x  1000. 


naked  eye  as  whitish  yellow  points, 
which  afterwards  become  more  dis- 
tinctly yellow.  Liquefaction  occurs 
around  these,  and  little  cups  are 
formed,  at  the  bottom  of  which  the 
colonies  form  little  yellowish  masses. 
On  agar,  a  stroke  culture  forms  a 
line  of  abundant  yellowish  growth, 
with  smooth,  shining  surface,  well 
formed  after  twenty-four  hours  at 
37°  C.  Later  it  becomes  bright 
orange  in  colour,  and  resembles  a 

streak  of  oil  paint.  Single  colonies  on  the  surface  of  agar  are 
circular  discs  of  similar  appearance,  which  may  reach  2  mm.  or  more 
in  diameter.  On  potatoes  it  grows  well  at  ordinary  temperature, 
forming  a  somewhat  abundant  layer  of  orange  colour.  In  bouillon 
it  produces  a  uniform  turbidity,  which  afterwards  settles  to  the 
bottom  as  an  abundant  layer,  which  assumes  a  brownish  yellow  tint. 
In  the  various  media  it  renders  the  reaction  acid,  and  it  coagulates 


FIG.  53.  —  Two  stab  cultures 
of  Staphylococcus  pyogenes 
aureus  in  gelatin,  (a)  10  days 
old,  (b)  3  weeks  old,  showing 
liquefaction  of  the  medium 
and  characters  of  growth. 
Natural  size. 


176         INFLAMMATION   AND   SUPPUKATION 

milk,  in  which  it  readily  grows.  The  cultures  have  a  somewhat 
sour  odour. 

It  has  considerable  tenacity  of  life  outside  the  body,  cultures 
in  gelatin  often  being  alive  after  having  been  kept  for  several 
months.  It  also  requires  a  rather  higher  temperature  to  kill 
it  than  most  spore-free  bacteria,  viz.  80°  C.  for  half  an  hour 
(Liibbert). 

The  staphylococcus  pyogenes  albus  is  similar  in  character, 
with  the  exception  that  its  growth  on  all  the  media  is  white. 
The  colour  of  the  staphylococcus  aureus  may  become  less  dis- 
tinctly yellow  after  being  kept  for  some  time  in  culture,  but  it 
never  assumes  the  white  colour  of  the  staphylococcus  albus,  and 
it  has  not  been  found  possible  to  transform  the  one  organism 
into  the  other.  A  micrococcus  called  by  Welch  staphylococcus 
epidermidis  albus  is  practically  always  present  in  the  skin 
epithelium ;  it  is  distinguished  by  its  relatively  non-pathogenic 
properties  and  by  liquefying  gelatin  somewhat  slowly.  It  is 
probably  an  attenuated  variety  of  the  staphylococcus  albus. 

The  staphylococcus  pyogenes  citreus,  which  is  less  frequently 
met  with,  differs  in  the  colour  of  the  cultures  being  a  lemon 

yellow,  and  is  less  virulent 
than  the  other  two. 

The  staphylococcus 
cereus  albus  and  staphy- 
lococcus cereus  flavus  are 
of  much  less  importance. 
They  produce  a  wax-like 
growth  on  gelatin  without 
liquefaction ;  hence  their 
name. 

Streptococcus  pyo- 
genes. —  This  organism 
is  a  coccus  of  slightly 

^H  larger      size     than     the 

(O^  staphylococcus      aureus 

-'•-..- .  ...»  about   1   p  in  diameter, 

FIG.  54,-Streptococciis  pyogenes,  young  cul-    and   forms   chfns  which 
tare  on  agar,  showing  chains  of  cocci.  may  contain  a  large  num- 

Stained  with  weak  carbol-fuchsin.     x  1000.      ber   of   members,   especi- 
ally when  it  is  growing 

in  fluids  (Fig.  54).  The  chains  vary  somewhat  in  length  in 
different  specimens,  and  on  this  ground  varieties  have  been  dis- 
tinguished, e.g.  the  streptococcus  brevis  and  streptococcus  longus 
(vide  infra).  As  division  may  take  place  in  many  of  the  cocci 


STREPTOCOCCUS   PYOGENES  177 

in  a  chain  at  the  same  time,  the  appearance  of  a  chain  of 
diplococci  is  often  met  with.  In  young  cultures  the  cocci  are 
fairly  uniform  in  size,  but  after  a  time  they  present  con- 
siderable variations,  many  swelling  up  to  twice  their  normal 
diameter.  These  are  to  be  regarded  as  involution  forms.  In 
its  staining  reactions  the  streptococcus  resembles  the  staphylo- 
cocci  described,  being  readily  coloured  by  Gram's  method. 

Cultivation. — In  cultures  outside  the  body  the  streptococcus 
pyogenes  grows  much  more  slowly  than  the  staphylococci,  and  also 


. . 

-VV  ' 


V 

~  » 


•  -f    -,"*• 

I  *»* 

FIG.    55.  —  Culture    of    the 
streptococcus  pyogenes  on 

an    agar    plate,    showing  FIG.  56. — Bacillus  pyocyaneus  ;  young 

numerous   colonies — three  culture  on  agar. 

successive  strokes.  Twenty-  Stained  with  weak  carbol-fuchsin.    x  1000. 

four  hours'  growth.    Natu- 
ral size. 

dies  out  more  readily,  being  in  every  respect  a  more  delicate 
organism. 

In  peptone  gelatin  a  stab  culture  shows,  about  the  second  day, 
a  thin  line,  which  in  its  subsequent  growth  is  seen  to  be  formed  of 
a  row  of  minute  rounded  colonies  of  whitish  colour,  which  may  be 
separate  at  the  lower  part  of  the  puncture.  They  do  not  usually 
exceed  the  size  of  a  small  pin's  head,  this  size  being  reached  about 
the  fifth  or  sixth  day.  The  growth  does  not  spread  on  the  surface, 
and  no  liquefaction  of  the  medium  occurs.  The  colonies  in  gelatin 
plates  have  a  corresponding  appearance,  being  minute  spherical 
points  of  whitish  colour.  A  somewhat  warm  temperature  is 
12 


178         INFLAMMATION   AND   SUPPURATION 

necessary  for  growth  j  even  at  20°  C.  some  varieties  do  not 
grow.  On  the  agar  media  growth  takes  place  along  the  stroke 
as  a  collection  of  small  circular  discs  of  semi -translucent 
appearance,  which  show  a  great  tendency  to  remain  separate 
(Fig.  55).  The  separate  colonies  remain  small,  rarely  exceeding 
1  mm.  in  diameter.  Cultures  on  agar  kept  at  the  body 
temperature  may  often  be  found  to  be  dead  after  ten  days.  On 
potato,  as  a  rule,  no  visible  growth  takes  place.  In  milk  it 
produces  a  strongly  acid  reaction  but  no  clotting  of  the  medium. 
It  ferments  lactose,  saccharose,  and  salicin  (Andrewes  and 
Horder) ;  it  produces  no  fermentation  of  inulin  in  Hiss's  serum- 
water-medium,  in  this  respect  differing  from  the  pneumococcus. 
It  has  a  strong  hsemolytic  action,  as  can  be  demonstrated  by 
growing  it  in  blood-agar  plates  (p.  38).  In  bouillon,  growth 
forms  numerous  minute  granules  which  afterwards  fall  to 
the  bottom,  the  deposit,  which  is  usually  not  very  abundant, 
having  a  sandy  appearance.  The  appearance  in  broth,  however, 
presents  variations  which  have  been  used  as  an  aid  to  distinguish 
different  species  of  streptococci.  It  has  been  found  that  those 
which  form  the  longest  chains  grow  most  distinctly  in  the  form  of 
spherical  granules,  those  forming  short  chains  giving  rise  to  a 
finer  deposit.  To  a  variety  which  forms  distinct  spherules  of 
minute  size  the  term  streptococcus  conglomeratus  has  been  given. 

Varieties  of  Streptococci. — Formerly  the  streptococcus  pyogenes 
and  the  streptococcus  erysipelatis  were  regarded  as  two  distinct 
species,  and  various  points  of  difference  between  them  were 
given.  Further  study,  and  especially  the  results  obtained  by 
modifying  the  virulence  (p.  182),  have  shown  that  these  dis- 
tinctions cannot  be  maintained,  and  now  practically  all  authorities 
are  agreed  that  the  two  organisms  are  one  and  the  same, 
erysipelas  being  produced  when  the  streptococcus  pyogenes  of  a 
certain  standard  of  virulence  gains  entrance  to  the  lymphatics  of 
the  skin.  Petruschky,  moreover,  showed  conclusively  by  inocu- 
lation that  a  streptococcus  cultivated  from  pus  could  cause 
erysipelas  in  the  human  subject. 

Streptococci  have  also  been  classified  according  to  the  length 
of  the  chains.  Thus  there  have  been  distinguished  (a)  strepto- 
coccus longus,  which  occurs  in  long  chains  and  is  pathogenic  to 
rabbits  and  mice ;  (b)  streptococcus  brevis,  which  is  common  in 
the  mouth  in  normal  conditions,  and  is  usually  non-pathogenic ; 
and  (c)  streptococcus  conglomeratus,  so  called  from  its  forming  in 
bouillon  minute  granules  composed  of  very  long  chains.  It  may 
be  stated  that  pathogenic  streptococci  obtained  from  the  human 
subject  usually  form  fairly  long  chains  on  agar,  whilst  the  short 


STREPTOCOCCUS   PYOGENES  179 

streptococci  obtained  from  the  mouth  and  intestine  are  usually 
devoid  of  virulence.  But  to  these  statements  exceptions  occur, 
as  short  streptococci  may  be  associated  with  grave  lesions ;  it 
has  also  been  found  that  the  length  of  the  chains  is  not  a 
constant  feature.  As  in  the  case  of  other  organisms  attempts 
have  also  been  made  to  differentiate  streptococci  by  means  of 
their  fermentative  properties.  Mervyn  Gordon  introduced  for 
this  purpose  nine  tests,  namely : — (1)  The  clotting  of  milk, 
(2)  the  reduction  of  neutral  red,  (3-9)  the  fermentation  with 
acid  production  of  saccharose,  lactose,  ramnose,  inulin,  salicin, 
coniferin,  and  mannite.  Andrewes  and  Horder  by  means  of 
these  have  differentiated  six  varieties,  of  which  five  occur  in  the 
human  subject.  These  are : — (a)  A  short-chained  form  called 
streptococcus  mitis,  which  occurs  chiefly  in  the  saliva  and  faeces 
as  a  saprophyte.  (6)  The  streptococcus  pyogenes,  which  is  the 
most  important  pathogenic  variety,  and  has  the  characters 
described  above,  (c)  The  streptococcus  salivarius,  which  corre- 
sponds to  the  streptococcus  brevis  of  the  mouth,  and  which,  as 
regards  fermentative  action,  seems  to  bear  the  same  relation  to 
the  next  variety  as  the  streptococcus  mitis  does  to  the  strepto- 
coccus pyogenes.  It  has  more  active  fermentative  properties 
and  clots  milk.  (cT)  The  streptococcus  anginosus,  which  corre- 
sponds with  the  so-called  streptococcus  scarlatinae  and  the  strepto- 
coccus conglomeratus.  It  usually  clots  milk  and  does  not  grow 
on  gelatin  at  20°  C.  (e)  The  streptococcus  fcecalis,  a  short- 
chained  form,  which  abounds  in  the  intestine  and  which  has 
great  fermentative  activity.  It  forms  sulphuretted  hydrogen, 
and  is  devoid  of  haemolytic  action.  (/)  The  sixth  variety  is  the 
streptococcus  equinus,  which  is  common  in  the  air  and  dust  of 
towns,  and  appears  to  be  derived  from  horse  dung.1 

Schottmuller  has  employed  the  appearance  of  the  colonies  of 
streptococci  on  blood  agar  as  a  means  of  separating  varieties, 
the  medium  used  consisting  of  two  parts  human  blood  and  five 
parts  melted  agar.  He  distinguishes  the  streptococcus  longus  or 
erysipelatis,  which  forms  grey  colonies  and  has  a  haemolytic 
action ;  a  streptococcus  mitior  or  viridans,  a  short  -  chained 
organism,  which  produces  small  green  colonies  and  very  little 
haemolysis,  and  a  streptococcus  mucosus  encapsulatus,  which,  as 
its  name  indicates,  shows  well -marked  capsules  and  produces 
colonies  which  have  a  slimy  consistence.  It  should  be  noted 
that  on  blood  agar  the  pneumococcus  forms  green  colonies  and 
produces  no  haemolysis. 

1  For  further  details  reference  must  be  made  to  the  original  papers,  Lancet, 
September  1906,  ii.  708,  etc. 


180         INFLAMMATION   AND   SUPPURATION 

It  will  be  thus  seen  from  this  account  that  the  streptococcus 
pyogenes  as  described  above  is  the  organism  most  frequently 
associated  with  the  pathogenic  processes,  and  that  short-chained 
forms  are  common  saprophytes  in  the  human  body,  although 
they  may  be  associated  with  conditions  of  disease ;  these  may 
be  subdivided  according  to  their  fermentative  activity  as 
detailed.  And  lastly,  there  is  the  streptococcus  conglomeratus 
(anginosus),  which  is  specially  abundant  in  the  throat  in  scarlet 
fever,  though  it  also  occurs  in  other  acute  catarrhal  states.  No 
definite  statement  can  yet  be  made  as  to  the  etiological  relation 
of  streptococci  to  scarlet  fever ;  we  can  only  say  that  streptococci 
are  almost  invariably  present  in  the  fauces,  and  that  to  them 
many  of  the  complications  of  the  disease  are  due. 

Bacillus  coli  communis. — The  microscopic  and  cultural  characters  are 
described  in  the  chapter  on  typhoid  fever.  The  bacillus  lactis  cerogenes 
and  the  bacillus  pyogenes  fcetidus  closely  resemble  it  ;  they  are  either 
varieties  or  closely  related  species.  The  former  is  distinguished  by 
producing  more  abundant  gas  formation,  and  by  its  growth  on  gelatin, 
etc.,  being  thicker  and  whiter  than  that  of  the  bacillus  coli. 

Bacillus  sere-genes  encapsulatus  sometimes  invades  the  tissues  before 
death,  and  is  characterised  by  the  formation  of  bubbles  of  gas  in  the 
infected  parts.  Its  characters  are  described  in  Chapter  XVI. 

Bacillus  pyocyaneus. — This  organism  occurs  in  the  form  of  minute 
rods  1-5  to  8  //,  in  length  and  less  than  '5  //,  in  thickness  (Fig.  56). 
Occasionally  two  or  three  are  found  attached  end  to  end.  They  are 
actively  motile,  and  do  not  form  spores.  They  stain  readily  with  the 
ordinary  basic  stains,  but  are  decolorised  by  Gram's  method. 

Cultivation. — It  grows  readily  on  all  the  ordinary  media  at  the  room 
temperature,  the  cultures  being  distinguished  by  the  formation  of  a 
greenish  pigment.  In  puncture  cultures  in  peptone-gelatin  a  greyish 
line  appears  in  twenty- four  hours,  and  at  its  upper  part  a  small  cup  of 
liquefaction  forms  within  forty-eight  hours.  At  this  time  a  slightly 
greenish  tint  is  seen  in  the  superficial  part  of  the  gelatin.  The 
liquefaction  extends  pretty  rapidly,  the  fluid  portion  being  turbid  and 
showing  masses  of  growth  at  its  lower  part.  The  green  colour 
becomes  more  and  more  marked  and  diffuses  through  the  gelatin. 
Ultimately  liquefaction  reaches  the  wall  of  the  tube.  In  plate  cultures 
the  colonies  appear  as  minute  whitish  points,  those  on  the  surface  being 
the  larger.  Under  a  low  power  of  the  microscope  they  have  a  brownish- 
yellow  colour  and  show  a  nodulated  surface,  the  superficial  colonies  being 
thinner  and  larger.  Liquefaction  soon  occurs,  the  colonies  on  the  surface 
forming  shallow  cups  with  small  irregular  masses  of  growth  at  the 
bottom,  the  deep  colonies  small  spheres  of  liquefaction.  Around  the 
colonies  a  greenish  tint  appears.  On  agar  the  growth  forms  an  abundant 
slimy  greyish  layer  which  afterwards  becomes  greenish,  and  a  bright 
green  colour  diffuses  through  the  whole  substance  of  the  medium.  On 
potatoes  the  growth  is  an  abundant  reddish-brown  layer  resembling  that 
of  the  glanders  bacillus,  and  the  potato  sometimes  shows  a  greenish 
discoloration. 

From  the  cultures  there  can  be  extracted  by  chloroform  a  coloured 
body  pyocyanin,  which  belongs  to  the  aromatic  series,  and  crystallises 


EXPERIMENTAL   INOCULATION  181 

in  the  form  of  long,  delicate  bluish-green  needles.     On  the  addition  of 
a  weak  acid  its  colour  changes  to  a  red. 

This  organism  has  distinct  pathogenic  action  in  certain  animals. 
Subcutaneous  injection  of  small  doses  in  rabbits  may  produce  a  local 
suppuration,  but  if  the  dose  be  large,  spreading  haemorrhagic  oedema 
results,  which  may  be  attended  by  septicaemia.  Intravenous  injection 
may  produce,  according  to  the  dose,  rapid  septicaemia  with  nephritis,  or 
sometimes  a  more  chronic  condition  of  wasting  attended  by  albuminuria. 

Micrococcus  tetragenus. 
— This  organism,  first  de- 
scribed by  Gaffky,  is  char- 
acterised by  the  fact  that  it 
divides  in  two  planes  at  right 
angles  to  one  another  (Fig.  +  fe» 
57),  and  is  thus  generally  *J  "  *  *  1 

found  in  the  tissues  in  groups  »  iJ  _f        *          W 

of  four  or  tetrads,  which  are     ££'-.  W       *  *      * 

often  seen  to  be  surrounded         ^ 

by   a    capsule.      The    cocci    ;  li 

measure    1    ^    in    diameter.     !  ^     M 

They  stain  readily  with  all 
the  ordinary  stains,  and  also 
retain  the  stain  in  Gram's 
method.  * 

It    grows    readily    on    all  *  tf> 

the  media  at  the  room  tern-  *     $F          * 

perature.  In  a  puncture  cul- 
ture on  peptone  -  gelatin  a 

Cm/alollfthetrack'of  "he      F'°-  57-Micrococcus  tetragenus  ;  young 
needle,  whilst  on  the  surface   c    ,..?^"  «S».  £?«•«. " 


-      W    ' 
i  * 


there  is  a  thick  rounded  disc   Stained  with  weak  carbol-fuchsin. 
of  whitish  colour.     The  gela- 
tin is  not  liquefied.     On  the  surface  of  agar  and  of  potato  the  growth 
is  an  abundant  moist  layer  of  the  same  colour.     The  growth  on  all 
the  media  has  a  peculiar  viscid  or  tenacious  character,  owing  to  the 
gelatinous  character  of  the  sheaths  of  the  cocci. 

White  mice  are  exceedingly  susceptible  to  this  organism.  Subcutaneous 
injection  is  followed  by  a  general  septicaemia,  the  organism  being  found 
in  large  numbers  in  the  blood  throughout  the  body.  Guinea-pigs  are 
less  susceptible  ;  sometimes  only  a  local  abscess  with  a  good  deal  of 
necrotic  change  results  ;  sometimes  there  is  also  septicaemia. 

Experimental  Inoculation. — We  shall  consider  chiefly  the 
staphylococcus  pyogenes  aureus  and  the  streptococcus  pyogenes, 
as  these  have  been  most  fully  studied. 

It  may  be  stated  at  the  outset  that  the  occurrence  of  suppura- 
tion depends  upon  the  number  of  organisms  introduced  into  the 
tissues,  the  number  necessary  varying  not  only  in  different 
animals,  but  also  in  different  parts  of  the  same  animal, — a  smaller 
number  producing  suppuration  in  the  anterior  chamber  of  the 
eye,  for  example,  than  in  the  peritoneum.  The  virulence  of  the 


182         IMFLAMMATION  AND   SUPPURATION 

organism  also  may  vary,  and  corresponding  results  may  be  pro- 
duced. Especially  is  this  so  in  the  case  of  the  streptococcus 
pyogenes. 

The  staphylococcus  aureus,  when  injected  subcutaneously  in 
suitable  numbers,  produces  an  acute  local  inflammation,  which 
is  followed  by  suppuration,  in  the  manner  described  above. 
The  spread  of  the  suppuration  goes  pari  passu  with  the  growth 
of  the  cocci.  If  a  large  dose  is  injected  the  cocci  may  enter  the 
blood  stream  in  sufficient  numbers  to  cause  secondary  suppurative 
foci  in  internal  organs  (cf.  intravenous  injection). 

Intravenous  injection  in  rabbits,  for  example,  produces  interest- 
ing results  which  vary  according  to  the  quantity  used.  If  a  con- 
siderable quantity  be  injected,  the  animal  may  die  in  twenty-four 
hours  of  a  general  septicaemia,  numerous  cocci  being  present  in 
the  capillaries  of  the  various  organs,  often  forming  plugs.  If  a 
smaller  quantity  be  used,  the  cocci  gradually  disappear  from  the 
circulating  blood ;  some  become  destroyed,  while  others  settle  in 
the  capillary  walls  in  various  parts  and  produce  minute  abscesses. 
These  are  most  common  in  the  kidneys,  where  they  occur  both 
in  the  cortex  and  medulla  as  minute  yellowish  areas  surrounded 
by  a  zone  of  intense  congestion  and  haemorrhage.  Similar  small 
abscesses  may  be  produced  in  the  heart  wall,  in  the  liver,  under 
the  periosteum,  and  in  the  interior  of  bones,  and  occasionally  in 
the  striped  muscles.  Very  rarely  indeed,  in  experimental 
injection,  do  the  cocci  settle  on  the  healthy  valves  of  the  heart. 
If,  however,  when  the  organisms  are  injected  into  the  blood, 
there  be  any  traumatism  of  a  valve,  or  of  any  other  part  of  the 
body,  they  show  a  special  tendency  to  settle  at  these  weakened 
points. 

Experiments  on  the  human  subject  have  also  proved  the 
pyogenic  properties  of  those  organisms.  Garre  inoculated 
scratches  near  the  root  of  his  finger-nail  with  a  pure  culture,  a 
small  cutaneous  pustule  resulting  ;  and  by  rubbing  a  culture  over 
the  skin  of  the  forearm  he  caused  a  carbuncular  condition  which 
healed  only  after  some  weeks.  Confirmatory  experiments  of 
this  nature  have  been  made  by  Bockhart,  Bumm,  and  others. 

When  tested  experimentally  the  staphylococcus  pyogenes  albus 
has  practically  the  same  pathogenic  effects  as  the  staphylococcus 
aureus. 

The  streptococcus  pyogenes  is  an  organism  the  virulence  of 
which  varies  much  according  to  the  diseased  condition  from  which 
it  has  been  obtained,  and  also  one  which  loses  its  virulence 
rapidly  in  cultures.  Even  highly  virulent  cultures,  if  grown 
under  ordinary  conditions,  in  the  course  of  time  lose  practically  all 


BACILLUS  COLI  COMMUNIS,  183 

pathogenic  power.  By  passage  from  animal  to  animal,  however, 
the  virulence  may  be  much  increased,  and  paripassu  the  effects  of 
inoculation  are  correspondingly  varied.  Marmorek,  for  example, 
found  that  the  virulence  of  a  streptococcus  can  be  enormously 
increased  by  growing  it  alternately  (a)  in  a  mixture  of  human 
blood  serum  and  bouillon  (vide  page  41),  and  (b)  in  the  body  of 
a  rabbit ;  ultimately,  after  several  passages  it  possesses  a  super- 
virulent  character,  so  that  even  an  extremely  minute  dose  intro- 
duced into  the  tissues  of  a  rabbit  produces  rapid  septicaemia,  with 
death  in  a  few  hours.  It  has  been  proved  by  Marmorek's  experi- 
ments, and  those  of  others,  that  the  same  species  of  streptococcus 
may  produce  at  one  time  merely  a  passing  local  redness,  at 
another  a  local  suppuration,  at  another  a  spreading  erysi- 
pelatous  condition,  or  again  a  general  septicaemic  infection, 
according  as  its  virulence  is  artificially  increased.  Such  experi- 
ments are  of  extreme  importance  as  explaining  to  some  extent  the 
great  diversity  of  lesions  in  the  human  subject  with  which  strep- 
tococci are  associated. 

Bacillus  Coli  Communis. — The  virulence  of  this  organism  also 
varies  much  and  can  be  increased  by  passage  from  animal  to 
animal.  Injection  into  the  serous  cavities  of  rabbits  produces  a 
fibrinous  inflammation  which  becomes  purulent  if  the  animal 
lives  sufficiently  long.  If,  however,  the  virulence  of  the  organism 
be  of  a  high  order,  death  takes  place  before  suppuration  is 
established,  and  there  is  a  septicaemic  condition,  the  organisms 
occurring  in  large  numbers  in  the  blood.  Intravenous  injection 
of  a  few  drops  of  a  virulent  bouillon  culture  usually  produces  a 
rapid  septicaemia  with  scattered  haemorrhages  in  various  organs. 

Other  Effects. — It  has  been  found  by  independent  observers  that  in 
cases  where  rabbits  recover  after  intravenous  injection  of  bacillus  coli 
communis,  a  certain  proportion  suifer  from  paralysis  and  sometimes  from 
atrophy  of  muscles,  especially  of  the  posterior  limbs,  these  symptoms 
being  due  to  lesions  of  the  cells  in  the  anterior  cornua  of  the  spinal  cord. 
Somewhat  similar  results  have  been  obtained  by  others  after  inoculations 
with  staphylococci  and  streptococci,  a  certain  proportion  only  of  the 
animals  showing  paralytic  symptoms  and  corresponding  changes  in  the 
spinal  cord.  The  lesions  are  believed  to  be  due  chiefly  to  the  action  of 
the  products  of  the  organisms  on  the  highly  organised  nervous  elements 

ch  further  research 


Much  further  research  requires  to  be  done  before  the  importance  of  these 
results  can  be  properly  estimated,  but  it  is  not  improbable  that  they 
will  throw  light  on  the  causation  of  nervous  lesions  which  occur  in  the 
human  subject,  and  the  etiology  of  which  at  present  is  quite  obscure. 
Some  observers,  chiefly  of  the  French  school,  consider  that  paralysis 
associated  with  cystitis,  in  which  the  bacillus  coli  communis  is  often 
present,  may  have  such  a  causation,  and  that  paralytic  conditions 
following  acute  infective  fevers  may  be  produced  by  the  products  of 
pyogenic  cocci,  which  frequently  occur  in  these  conditions. 


184         INFLAMMATION  AND   SUPPURATION 

Lesions  in  the  Human  Subject.  —  The  following  statement 
may  be  made  with  regard  to  the  occurrence  of  the  chief  organisms 
mentioned,  in  the  various  suppurative  and  inflammatory  con- 
ditions in  the  human  subject.  The  account  is,  however,  by  no 
means  exhaustive,  as  clinical  bacteriology  has  shown  that  practi- 
cally every  part  of  the  body  may  be  the  site  of  a  lesion  produced 
by  the  pyogenic  bacteria.  It  may  also  be  noted  that  acute 
catarrhal  conditions  of  cavities  or  tubes  are  in  many  cases  also 
to  be  ascribed  to  their  presence. 

The  staphylococci  are  the  most  common  causal  agents  in 
localised  abscesses,  in  pustules  on  the  skin,  in  carbuncles,  boils, 

etc.,  in  acute  suppurative 
periostitis,  in  catarrhs  of 
mucous  surfaces,  in  ulcer- 
ative  endocarditis,  and  in 
various  pysemic  conditions. 
They  may  also  be  present 
in  septicaemia. 

Streptococci  are  especi- 
ally  found  in  spreading 
inflammation  with  or  with- 
out  suppuration  ;  in  diffuse 

1B^  %j$j      phlegmonous    and    erysi- 

pelatous  conditions,  sup- 
purations in  serous  mem- 
branes and  in  joints  (Fig. 
58).  They  also  occur  in 

PIG.  58.-Streptococci  in  acute  suppuration.   aCUte    suppurative.    perio- 

Corrosive  film  ;  stained  by  Gram's  method     stltis    and    ulcerative    en- 

and  safranin.      x  1000.  docarditis.  Secondary 

abscesses     in     lymphatic 

glands  and  lymphangitis  are  also,  we  believe,  more  frequently 
caused  by  streptococci  than  staphylococci.  They  also  produce 
nbrinous  exudation  on  the  mucous  surfaces,  leading  to  the 
formation  of  false  membrane  in  many  of  the  cases  of  non- 
diphtheritic  inflammation  of  the  throat,  which  are  met  with  in 
scarlatina  l  and  other  conditions,  and  they  are  also  the  organisms 
most  frequently  present  in  acute  catarrhal  inflammations  in  this 
situation.  In  puerperal  peritonitis  they  are  frequently  found  in 
a  condition  of  purity,  and  they  also  appear  to  be  the  most 
frequent  cause  of  puerperal  septicaemia,  in  which  condition  they 
may  be  found  after  death  in  the  capillaries  of  various  organs. 

1  True  diphtheria  may  also  occasionally  be  associated  with  this  disease, 
usually  as  a  sequel. 


I 
-  I 


LESIONS   IN   THE   HUMAN   SUBJECT         185 

In  pyaemia  they  are  frequently  present,  though  in  most  cases 
associated  with  other  pyogenic  organisms.  Some  cases  of 
enteritis  in  infants — streptococcic  enteritis — are  also  apparently 
due  to  a  streptococcus,  which,  however,  presents  in  cultures 
certain  points  of  difference  from  the  streptococcus  pyogenes. 

The  bacillus  coli  communis  is  found  in  a  great  many  inflam- 
matory and  suppurative  conditions  in  connection  with  the  ali- 
mentary tract — for  example,  in  suppuration  in  the  peritoneum, 
or  in  the  extraperitoneal  tissue  with  or  without  perforation  of  the 
bowel,  in  the  peritonitis  following  strangulation  of  the  bowel,  in 
appendicitis  and  the  lesions  following  it,  in  suppuration  around 
the  bile-ducts,  etc.  It  may  also  occur  in  lesions  in  other  parts 
of  the  body, — endocarditis,  pleurisy,  etc.,  which  in  some  cases 
are  associated  with  lesions  of  the  intestine,  though  in  others  such 
cannot  be  found.  It  is  also  frequently  present  in  inflammation 
of  the  urinary  passages,  cystitis,  pyelitis,  abscesses  in  the  kidneys, 
etc.,  these  lesions  being  in  fact  most  frequently  caused  by  this 
or  closely  allied  organisms. 

In  certain  cases  o£  enteritis  it  is  probably  the  causal  agent, 
though  this  is  difficult  of  proof,  as  it  is  much  increased  in 
numbers  in  practically  all  abnormal  conditions  of  the  intestine. 
We  may  remark  that  it  has  been  repeatedly  proved  that  the 
bacillus  coli  cultivated  from  various  lesions  is  more  virulent  than 
that  in  the  intestine,  its  virulence  having  been  heightened  by 
growth  .in  the  tissues. 

The  micrococcus  tetragenus  is  often  found  in  suppurations  in 
the  region  of  the  mouth  or  in  the  neck,  and  also  occurs  in 
various  lesions  of  the  respiratory  tract,  in  phthisical  cavities, 
abscesses  in  the  lungs,  etc.  Sometimes  it  is  present  alone,  and 
probably  has  a  pyogenic  action  in  the  human  subject  under 
certain  conditions.  In  other  cases  it  is  associated  with  other 
organisms.  Recently  one  or  two  cases  of  pyaemia  have  been 
described  in  which  this  organism  was  found  in  a  state  of  purity 
in  the  pus  in  various  situations.  In  this  latter  condition  the  pus 
has  been  described  as  possessing  an  oily,  viscous  character,  and 
as  being  often  blood-stained. 

The  bacillus  pyocyaneus  is  rarely  found  alone  in  pus,  though 
it  is  not  infrequent  along  with  other  organisms.  We  have  met 
with  it  twice  in  cases  of  multiple  abscesses,  in  association  with 
the  staphylococcus  pyogenes  aureus.  Lately  some  diseases  in 
children  have  been  described  in  which  the  bacillus  pyocyaneus 
has  been  found  throughout  the  body ;  in  these  cases  the  chief 
symptoms  have  been  fever,  gastro-intestinal  irritation,  pustular 
or  petechial  eruptions  on  the  skin,  and  general  marasmus. 


186         INFLAMMATION   AND   SUPPURATION 

Suppurative  and  inflammatory  conditions,  associated  with  the 
organisms  of  special  diseases,  will  be  described  in  the  respective 
chapters. 

Mode  of  Entrance  and  Spread. — Many  of  the  organisms  of 
suppuration  have  a  wide  distribution  in  nature,  and  many  also 
are  present  on  the  skin  and  mucous  membranes  of  healthy 
individuals.  Staphylococci  are  commonly  present  on  the  skin, 


FIG.   59. — Minute  focus  of  commencing  suppuration  in  brain — case  of  acute 
ulcerative   endocarditis.     In  the  centre  a  small  haemorrhage  ;   to  right 
side  dark  masses  of  staphylococci ;  zone  of  leucocytes  at  periphery. 
Alum  carmine  and  Gram's  method,      x  50. 

and  also  occur  in  the  throat  and  other  parts,  and  streptococci 
can  often  be  cultivated  from  the  secretions  of  the  mouth  in 
normal  conditions.  The  pneumococcus  of  Fraenkel  and  the 
pneumobacillus  of  Friedlander  have  also  been  found  in  the 
mouth  and  in  the  nasal  cavity,  whilst  the  bacillus  coli  communis 
is  a  normal  inhabitant  of  the  intestinal  tract.  The  entrance  of 
these  organisms  into  the  deeper  tissues  when  a  surface  lesion 
occurs  can  be  readily  understood.  Their  action  will,  of  course, 
be  favoured  by  any  condition  of  depressed  vitality.  Though  in 
normal  conditions  the  blood  is  bacterium-free,  we  must  suppose 


ENTRANCE  AND   SPREAD   OF   BACTERIA     187 

that  from  time  to  time  a  certain  number  of  such  organisms  gain 
entrance  to  it  from  trifling  lesions  of  the  skin  or  mucous  surfaces, 
the  possibilities  of  entrance  from  the  latter  being  especially 
numerous.  In  most  cases  they  are  killed  by  the  action  of  the 
healthy  serum  or  cells  of  the  body,  and  no  harm  results.  If, 
however,  there  be  a  local  weakness,  they  may  settle  in  that  part 


FIG.  60. — Secondary  infection  of  a  glomerulus  of  kidney  by  the  staphylo- 

coccus  aureus,  in  a  case  of  ulcerative  endocarditis.     The  cocci  (stained 

darkly)  are   seen  plugging   the  capillaries  and  also  lying  free.      The 

glomerulus  is  much  swollen,  infiltrated  by  leucocytes,  and  partly  necrosed. 

Paraffin  section  ;  stained  by  Gram's  method  and  Bismarck-brown,      x  300. 

and  produce  suppuration,  and  from  this  other  parts  of  the  body 
may  be  infected.  Such  a  supposition  as  this  is  necessary  to 
explain  many  inflammatory  and  suppurative  conditions  met  with 
clinically.  In  some  cases  of  multiple  suppurations  due  to 
staphylococcus  infection,  which  we  have  had  the  opportunity  to 
examine,  only  an  apparently  unimportant  surface  lesion  was 
present ;  whilst  in  others  no  lesion  could  be  found  to  explain 
the  origin  of  the  infection.  The  term  cryptogenetic  has  been 
applied  by  some  writers  to  such  cases  in  which  the  original 


188         INFLAMMATION  AND  SUPPUBATION 

point  of  infection  cannot  be  found,  but  its  use  is  scarcely 
necessary. 

The  paths  of  secondary  infection  may  be  conveniently  sum- 
marised thus  :  First,  by  lymphatics.  In  this  way  the  lymphatic 
glands  may  be  infected,  and  also  serous  sacs  in  relation  to  the 
organs  where  the  primary  lesion  exists.  Second,  by  natural 
channels,  such  as  the  ureters  and  the  bile-ducts,  the  spread 
being  generally  associated  with  an  inflammatory  condition  of  the 
lining  epithelium.  In  this  way  the  kidneys  and  liver  respectively 
may  be  infected.  Third,  by  the  blood  vessels :  (a)  by  a  few 
organisms  gaining  entrance  to  the  blood  from  a  local  lesion,  and 
settling  in  a  favourable  nidus  or  a  damaged  tissue,  the  original 
path  of  infection  often  being  obscure ;  (b)  by  a  septic  phlebitis 
with  suppurative  softening  of  the  thrombus  and  resulting  em- 
bolism ;  and  we  may  add  (c)  by  a  direct  extension  along  a  vein, 
producing  a  spreading  thrombosis  and  suppuration  within  the 
vein.  In  this  way  suppuration  may  spread  along  the  portal  vein 
to  the  liver  from  a  lesion  in  the  alimentary  canal,  the  condition 
being  known  as  pylephlebitis  suppurativa. 

Although  many  of  the  lesions  produced  by  the  bacteria 
under  consideration  have  already  been  mentioned,  certain  con- 
ditions may  be  selected  for  further  consideration  on  account  of 
their  clinical  importance  or  bacteriological  interest. 

Endocarditis. — There  is  now  strong  presumptive  evidence 
that  all  cases  of  endocarditis  are  due  to  bacterial  infection.  In 
the  simple  or  vegetative  form,  so  often  the  result  of  acute 
rheumatism,  the  micrococcus  rheumaticus  (p.  193)  has  been 
cultivated  from  the  valves  in  a  certain  number  of  cases,  and  is 
probably  the  causal  agent  in  most  instances. 

Endocarditis  of  the  ulcerative  type  may  be  produced  by 
various  organisms,  chiefly  pyogenic.  Of  these  the  staphylococci 
and  streptococci  are  most  frequently  found.  In  some  cases  of 
ulcerative  endocarditis  following  pneumonia,  the  pneumococcus 
(Fraenkel's)  is  present ;  in  others  pyogenic  cocci,  especially 
streptococci.  Other  organisms  have  been  cultivated  from 
different  cases  of  the  disease,  and  some  of  these  have  received 
special  names ;  for  example,  the  diplococcus  endocarditis  encap- 
sulatus,  bacillus  endocarditidis  griseus  (Weichselbaum),  and 
others.  In  some  cases  the  bacillus  coli  communis  has  been  found, 
and  occasionally  in  endocarditis  following  typhoid  the  typhoid 
bacillus  has  been  described  as  the  organism  present,  but  further 
observations  on  this  point  are  desirable.  The  gonococcus  also 
has  been  shown  to  affect  the  heart  valves  (p.  225),  though  this  is 
a  very  rare  occurrence.  Tubercle  nodules  on  the  heart  valves 


ENDOCARDITIS 


189 


have  been  found  in  a  few  cases  of  acute  tuberculosis,  though  no 
vegetative  or  ulcerative  condition  is  produced. 

In  some  cases,  though  we  believe  not  often,  the  organisms 
may  attack  healthy  valves,  producing  a  primary  ulcerative  endo- 
carditis, but  more  frequently  the  valves  have  been  the  seat  of 
previous  endocarditis,  secondary  ulcerative  endocarditis  being 


FIG.  61. — Section  of  a  vegetation  in';ulcerative  endocarditis,  showing  numerous 
staphylococci  lying  in  the  spaces.  The  lower  portion  is  a  fragment 
in  process  of  separation. 

Stained  by  Gram's  method  and  Bismarck-brown.      x  600. 

thus  produced.  In  some  cases,  especially  when  the  valves  have 
been  previously  diseased,  the  source  of  the  infection  is  quite 
obscure.  It  is  evident  that  as  the  vegetations  are  composed  for 
the  most  part  of  unorganised  material,  they  do  not  offer  the 
same  resistance  to  the  growth  of  bacteria,  when  a  few  reach  them, 
as  a  healthy  cellular  tissue  does.  On  microscopic  examination 
of  the  diseased  valves  the  organisms  are  usually  to  be  found  in 
enormous  numbers,  sometimes  forming  an  almost  continuous  layer 
on  the  surface,  or  occurring  in  large  masses  or  clusters  in  spaces 
in  the  vegetation  (Fig.  61).  By  their  action  a  certain  amount 


190         INFLAMMATION  AND   SUPPURATION 

of  softening  or  breaking  down  of  the  vegetations  occurs,  and  the 
emboli  thus  produced  act  as  the  carriers  of  infection  to  other 
organs,  and  give  rise  to  secondary  suppurations. 

Experimental. — Occasionally  ulcerative  endocarditis  is  produced  by  the 
simple  intravenous  injection  of  staphylococci  and  streptococci  into  the 
circulation,  but  this  is  a  very  rare  occurrence.  It  often  follows,  however, 
when  the  valves  have  been  previously  injured.  Orth  and  Wyssokowitsch 
at  a  comparatively  early  date  produced  the  condition  by  damaging  the 
aortic  cusps  by  a  glass  rod  introduced  through  the  carotid,  and  after- 
wards injecting  staphylococci  into  the  circulation.  Similar  experiments 
have  since  been  repeated  with  streptococci,  pneumococci,  and  other 
organisms,  with  like  result.  Blbbert  found  that  if  a  potato  culture  of 
the  staphylococcus  aureus  were  rubbed  down  in  salt  solution  so  as  to 
form  an  emulsion,  and  then  injected  into  the  circulation, ^some  minute 
fragments  became  arrested  at  the  attachment  of  the  chordae  tendinese  and 
produced  an  ulcerative  endocarditis. 

Acute  Suppuratiye  Periostitis  and  Osteomyelitis. — Special 
mention  is  made  of  this  condition  on  account  of  its  comparative 
frequency  and  gravity.  The  great  majority  of  cases  are  caused 
by  the  pyogenic  cocci,  of  which  one  or  two  varieties  may  be 
present,  the  staphylococcus  aureus,  however,  occurring  most 
frequently.  Pneumococci  have  been  found  alone  in  some  cases, 
and  in  a  few  cases  following  typhoid  fever,  apparently  well 
authenticated,  the  typhoid  bacillus  has  been  found  alone.  In 
others  again  the  bacillus  coli  communis  is  present. 

The  affection  of  the  periosteum  or  interior  of  the  bones  by 
these  organisms,  which  is  especially  common  in  young  subjects, 
may  take  place  in  the  course  of  other  affections  produced  by 
the  same  organisms  or  in  the  course  of  infective  fevers,  but  in  a 
great  many  cases  the  path  of  entrance  cannot  be  determined. 
In  the  course  of  this  disease  serious  secondary  infections  are 
always  very  liable  to  follow,  such  as  small  abscesses  in  the 
kidneys,  heart- wall,  lungs,  liver,  etc.,  suppurations  in  serous 
cavities,  and  ulcerative  endocarditis ;  in  fact,  some  cases  present 
the  most  typical  examples  of  extreme  general  staphylococcus 
infection.  The  entrance  of  the  organisms  into  the  blood  stream 
from  the  lesion  of  the  bone  is  especially  favoured  by  the  arrange- 
ment of  the  veins  in  the  bone  and  marrow. 

Experimental. — Multiple  abscesses  in  the  bones  and  under  the  peri- 
osteum may  occur  in  simple  intravenous  injection  of  the  pyogenic 
cocci  into  the  blood,  and  are  especially  liable  to  be  formed  when  young 
animals  are  used.  These  abscesses  are  of  small  size,  and  do  not  spread 
in  the  same  way  as  in  the  natural  disease  in  the  human  subject. 

In  experiments  on  healthy  animals,  however,  the  conditions  are  not 
analogous  to  those  of  the  natural  disease.  "We  must  presume  that  in  the 
latter  there  is  some  local  weakness  or  susceptibility  which  enables  the 


CONJUNCTIVITIS  191 

few  organisms  which  have  reached  the  part  by  the  blood  to  settle  and 
multiply.  Moreover,  if  a  bone  be  experimentally  injured,  e.g.  by  actual 
fracture  or  by  stripping  off  the  periosteum,  before  the  organisms  are 
injected,  then  a  much  more  extensive  suppuration  occurs  at  the  injured 
part. 

Erysipelas. — A  spreading  inflammatory  condition  of  the 
skin  may  be  produced  by  a  variety  of  organisms,  but  the  disease 
in  the  human  subject  in  its  characteristic  form  is  almost  in- 
variably due  to  a  streptococcus,  as  was  shown  by  Fehleisen  in 
1884.  He  obtained  pure  cultures  of  the  organism,  and  gave  it 
the  name  of  streptococcus  erysipelatis ;  and,  further,  by  inocu- 
lations on  the  human  subject  as  a  therapeutic  measure  in 
malignant  disease,  he  was  able  to  reproduce  erysipelas.  As 
stated  above,  however,  one  after  another  of  the  supposed  points 
of  difference  between  the  streptococcus  of  erysipelas  and  that  of 
suppuration  has  broken  down,  and  it  is  now  generally  held  that 
erysipelas  is  produced  by  the  streptococcus  pyogenes  of  a  certain 
degree  of  virulence.  It  must  be  noted,  however,  that  erysipelas 
passes  from  patient  to  patient  as  erysipelas,  and  purulent  con- 
ditions due  to  streptococci  do  not  appear  liable  to  be  followed 
by  erysipelas.  On  the  other  hand,  the  connection  between 
erysipelas  and  puerperal  septicaemia  is  well  established  clinically. 

In  a  case  of  erysipelas  the  streptococci  are  found  in  large 
numbers  in  the  lymphatics  of  the  cutis  and  underlying  tissues, 
just  beyond  the  swollen  margin  of  the  inflammatory  area.  As 
the  inflammation  advances  they  gradually  die  out,  and  after  a 
time  their  extension  at  the  periphery  comes  to  an  end.  The 
streptococci  may  extend  to  serous  and  synovial  cavities  and  set 
up  inflammatory  or  suppurative  change, — peritonitis,  meningitis, 
and  synovitis  may  thus  be  produced. 

Conjunctivitis. — A  considerable  number  of  organisms  are 
concerned  in  the  production  of  conjunctivitis  and  its  associated 
lesions.  Of  these  a  number  appear  to  be  specially  associated 
with  this  region.  Thus  a  small  organism,  generally  known  as 
the  Koch- Weeks  bacillus,  is  the  most  common  cause  of  acute 
contagious  conjunctivitis,  especially  prevalent  in  Egypt,  but 
also  common  in  this  country.  This  organism  is  very  minute, 
being  little  more  than  1  /*  in  length,  and  morphologically 
resembles  the  influenza  bacillus ;  its  conditions  of  growth  are 
even  more  restricted,  as  it  rarely  grows  on  blood  agar,  the  best 
medium  being  serum  agar.  On  this  medium  it  produces  minute 
transparent  colonies  like  drops  of  dew.  The  obtaining  of  pure 
cultures  is  a  matter  of  considerable  difficulty,  and  it  is  nearly 
always  accompanied  by  the  xerosis  bacillus.  It  can  readily  be 


192 


INFLAMMATION   AND   SUPPURATION 


found  in   the  muco-purulent  secretion  by  staining   films  with 

_  weak     (1  :  10)     carbol- 

fuchsin,  and  is  often  to 

be  seen   in  the   interior 

8^£  of   leucocytes  (Fig.    62). 

\       Another     organism     ex- 

^      ceedingly  like  the  prev- 

ious, apparently  differing 

JJJfZffZf  from  it  only  in  the  rather 

VM>*  wider    conditions    of 

growth,  is  Muller's  bacil- 
lus. It  has  been  culti- 
vated by  him  in  a  con- 
siderable proportion  of 
cases  of  trachoma,  but 
its  relation  to  this  con- 

FiG.62.-Film    preparation   from   a   case   of    djtion  is   sti11   matter   pf 

acute  conjunctivitis,  showing  Koch-  Weeks    dispute.      Another  bacil- 

bacilli,  chiefly  contained  within  a  leucocyte,    lus    which    is    now   well 

(From  a  preparation  by  Dr.  Inglis  Pollock.)     recognised   is    the   diplo- 

bacillus  of  conjunctivitis 

first  described  by  Morax.     It  is  especially  common  in  the  more 

subacute  cases  of  conjunctivitis.     Eyre  found  it  in  2  '5  per  cent 

of  all  cases  of   conjunc- 

tivitis.   Its  cultural  char- 

acters are  given   below. 

The      xerosis      bacillus, 

which   is  a  small  diph- 

theroid    organism    (Fig. 

123),  has  been  found  in 

xerosis    of   the  conjunc- 

tiva,   in    follicular   con- 

junctivitis, and  in  other 

conditions  ;     it    appears 

to  occur  sometimes  also 

in    the  normal   conjunc- 

tiva.     It     is     doubtful 

whether      it      has     any 

pathogenic  action  of  im- 

portance.       Acute     con- 

junctivitis     IS    also    pro 

duced   by   the    pneumo- 

coccus,  epidemics  of  the 

disease   being    sometimes   due  to   this   organism,  and  also  by 


63_Film  preparation  of  corijunctival 
secretion  showing  the  Morax  diplo-bacillus 
of  conjunctivitis.  xlOOO. 


ACUTE   RHEUMATISM  193 

streptococci  and  staphylococci.  True  diphtheria  of  the  con- 
junctiva caused  by  the  Klebs-Lofner  bacillus  also  occurs, 
whilst  in  gonorrhceal  conjunctivitis,  often  of  an  acute  purulent 
type,  the  gonococcus  is  present  (p.  225). 

Diplo  -  bacillus  of  Conjunctivitis. — This  organism,  discovered  by 
Morax,  is  a  small  plump  bacillus,  measuring  1  x  2  /i,  and  usually  occur- 
ring in  pairs,  or  in  short  chains  of  pairs  (Fig.  63).  It  is  non-motile, 
does  not  form  spores,  and  is  decolorised  by  Gram's  method.  It  does  not 
grow  on  the  ordinary  gelatin  and  agar  media,  the  addition  of  blood  or 
serum  being  necessary.  On  serum  it  forms  small  rounded  colonies 
which  produce  small  pits  of  liquefaction  ;  hence  it  has  been  called  the 
bacillus  lacunatus.  In  cultures  it  is  distinctly  pleomorphous,  and 
involution  forms  also  occur.  It  is  non- pathogenic  to  the  lower  animals. 

Acute  Rheumatism. — There  are  many  facts  which  point  to 
the  infective  nature  of  this  disease,  and  investigations  from  this 
point  of  view  have  yielded  important  results.  Of  the  organism 
isolated,  the  one  which  appears  to  have  strongest  claims  is  a 
small  coccus  observed  by  Triboulet,  and  by  Westphal  and 
Wassermann,  the  characters  and  action  of  which  were  first 
investigated  in  this  country  by  Poynton  and  Paine.  It  is  now 
usually  spoken  of  as  the  micrococcus  rheumaticus.  The  organism 
is  sometimes  spoken  of  as  a  diplococcus,  but  it  is  best  described 
as  a  streptococcus  growing  in  short  chains ;  in  the  tissues,  how- 
ever, it  usually  occurs  in  pairs.  It  is  rather  smaller  than  the 
streptococcus  pyogenes,  and  although  it  can  be  stained  by  Gram's 
method,  it  loses  the  colour  more  readily  than  the  streptococcus. 
In  the  various  media  it  produces  a  large  amount  of  acid,  and 
usually  clots  milk  after  incubation  for  two  days ;  on  blood  agar 
it  alters  the  haemoglobin  to  a  brownish  colour.  Its  growth  on 
media  generally  is  more  luxuriant  than  that  of  the  streptococcus, 
and  it  grows  well  on  gelatin  at  20°  C.  Intravenous  injection 
of  pure  cultures  in  rabbits  often  produces  polyarthritis  and 
synovitis,  valvulitis  and  pericarditis,  without  any  suppurative 
change — lesions  which  it  is  stated  are  not  produced  by  the 
ordinary  streptococci  (Beattie).  In  one  or  two  instances 
choreiform  movements  have  been  observed  after  injection.  The 
organism  is  most  easily  obtained  from  the  substance  of  inflamed 
synovial  membrane  where  it  is  invading  the  tissues;  a  part 
where  there  is  special  congestion  should  be  selected  as  being 
most  likely  to  give  positive  results.  It  is  only  occasionally  to 
be  obtained  from  the  fluid  in  joints.  It  has  also  been  cultivated 
from  the  blood  in  rheumatic  fever,  from  the  vegetations  on  the 
heart  valves,  and  from  other  acute  lesions ;  in  many  cases,  how- 
ever, cultures  from  the  blood  give  negative  results.  Poynton 
13 


194          INFLAMMATION   AND   SUPPURATION 

and  Paine  cultivated  it  from  the  cerebro-  spinal  fluid  in  three 
cases  where  chorea  was  present,  and  also  detected  it  in  the 
membranes  of  the  brain.  They  consider  that  this  disease  is 
probably  of  the  nature  of  a  slight  meningo-myelitis  produced  by 
this  organism.  The  facts  already  accumulated  speak  strongly 
in  favour  of  this  organism  being  causally  related  to  rheumatic 
fever,  though  this  cannot  be  considered  completely  proved. 
Andrewes  finds  that  the  organism  has  the  same  cultural  characters 
and  fermentative  effects  as  the  streptococcus  fsecalis,  a  common 
inhabitant  of  the  intestine.  Even,  however,  if  the  two  organisms 
were  the  same,  it  might  well  be  possible  that  rheumatic  fever  is 
due  to  an  infection  of  the  tissues  by  this  variety  of  streptococcus. 
The  clinical  data,  in  fact,  rather  point  to  rheumatic  fever  being 
due  to  an  infection  by  some  organism  frequently  present  in  the 
body,  brought  about  by  some  state  of  predisposition  or  acquired 
susceptibility. 

Vaccination  Treatment  of  Infections  by  the  Pyogenic  Cocci. 
— From  his  study  of  the  part  played  by  phagocytosis  in  the 
successful  combat  of  the  pyogenic  bacteria  by  the  body,  Wright 
was  led  to  advocate  the  treatment  of  such  infections  by  the 
origination  during  their  course  of  an  active  immunisation  by 
dead  cultures  of  the  infecting  agent.  The  treatment  is  applicable 
when  the  infection  is  practically  local  as  in  acne  pustules,  in  boils, 
etc.  (For  the  theoretical  questions  raised  see  Immunity.)  It 
is  best  to  attempt  to  isolate  the  causal  organism  from  the  lesion 
and  to  test  the  opsoriic  index  of  the  patient  against  it.  To 
prepare  the  vaccine  an  agar  slope  culture  is  taken  and  the 
growth  washed  off  with  normal  saline.  The  organism  is  then 
killed  by  steaming  for  an  appropriate  time,  and  the  efficacy  of 
the  sterilisation  tested  by  inoculating  fresh  agar  tubes.  The 
strength  of  the  emulsion  is  estimated  by  the  method  of  counting 
dead  bacteria  described  on  p.  67.  The  number  of  bacteria  used 
for  an  injection  is  from  250,000,000  to  500,000,000,  and  in  the 
details  of  the  measurement  of  this  quantity  and  in  its  injection 
every  aseptic  precaution  must,  of  course,  be  adopted.  If 
repeated  injections  are  necessary  Wright  recommends  that  the 
opsonic  index  should  be  observed  every  few  days  and  the 
injections  only  practised  during  a  positive  phase.  If  it  is  not 
practicable  to  use  the  infecting  strain  for  the  preparation  of  the 
vaccine,  then  laboratory  cultures  must  be  used,  and  in  such 
cases  it  is  well  to  use  a  mixture  of  strains ;  in  skin  infections 
a  mixture  of  staphylococcus  aureus  and  albus  may  be  employed. 
Such  means  have  been  extensively  used  in  the  treatment  of 
acne,  boils,  sycosis,  infections  of  the  genito-urinary  tract  by  the 


METHODS   OF   EXAMINATION  195 

b.  coli,  infections  of  joints  by  the  gonococcus,  and  in  many  cases 
considerable  success  has  followed  the  treatment. 

Methods  of  Examination  in  Inflammatory  and  Suppurative 
Conditions. — These  are  usually  of  a  comparatively  simple  nature, 
and  include  (1)  microscopic  examination,  (2)  the  making  of 
cultures. 

(1)  The  pus  or  other  fluids  should  be  examined  microscopi- 
cally,  first  of   all   by  means  of   film   preparations  in  order  to 
determine  the  characters  of  the  organisms  present.     The  films 
should  be  stained  (a)  by  one  of  the  ordinary  solutions,  such 
as  carbol-thionin-blue  (p.  98),  or  a  saturated  watery  solution  of 
methylene-blue ;   and  (6)  by  Gram's  method.     The  use  of  the 
latter  is  of  course  of  high  importance  as  an  aid  in  the  recognition. 

(2)  As  most  of  the  pyogenic  organisms  grow  readily  on  the 
gelatin  media  at  ordinary  temperatures,  pure  cultures  can  be 
readily  obtained  by  the  ordinary  plate  methods.     But  in  many 
cases  the  separation  can  be  effected  much  more  rapidly  by  the 
method  of  successive  streaks   on  agar  tubes,   which  are   then 
incubated  at  37°  C.      When  the  presence  of  pneumococci  is 
suspected  this  method  ought  always  to  be  used,  and  it  is  also  to 
be  preferred  in  the  case  of  streptococci.     Inoculation  experiments 
may  be  carried  out  as  occasion  arises. 

In  cases  of  suspected  blood  infection  the  examination  of  the 
blood  is  to  be  carried  out  by  the  methods  already  described 
(p.  68), 


CHAPTER   VII. 

INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS, 
CONTINUED:  THE  ACUTE  PNEUMONIAS,  EPI- 
DEMIC CEREBRO-SPINAL  MENINGITIS. 

Introductory. — The  term  Pneumonia  is  applied  to  several  con- 
ditions which  present  differences  in  pathological  anatomy  and  in 
origin.  All  of  these,  however,  must  be  looked  on  as  varieties  of 
inflammation  in  which  the  process  is  modified  in  different  ways 
depending  on  the  special  structure  of  the  lung  or  of  the  parts 
which  compose  it.  There  is,  first  of  all,  and,  in  adults,  the  com- 
monest type,  the  acute  croupous  or  lobar  pneumonia,  in  which 
an  inflammatory  process  attended  by  abundant  fibrinous  exuda- 
tion affects,  by  continuity,  the  entire  tissue  of  a  lobe  or  of  a 
large  portion  of  the  lung.  It  departs  from  the  course  of  an 
ordinary  inflammation  in  that  the  reaction  of  the  connective 
tissue  of  the  lung  is  relatively  slight,  and  there  is  usually  no 
tendency  for  organisation  of  the  inflammatory  exudation  to  take 
place.  Secondly,  there  is  the  acute  catarrhal  or  lobular 
pneumonia,  where  a  catarrhal  inflammatory  process  spreads  from 
the  capillary  bronchi  to  the  air  vesicles,  and  in  these  a  change, 
consisting  largely  of  proliferation  of  the  endothelium  of  the 
alveoli,  takes  place  which  leads  to  consolidation  of  patches  of 
the  lung  tissue.  Up  till  1889  acute  catarrhal  pneumonia  was 
comparatively  rare  except  in  children.  In  adults  it  was  chiefly 
found  as  a  secondary  complication  to  some  condition  such  as 
diphtheria,  typhoid  fever,  etc.  Since,  however,  influenza  in  an 
epidemic  form  has  become  frequent,  catarrhal  pneumonia  has 
been  of  much  more  frequent  occurrence  in  adults,  has  assumed 
a  very  fatal  tendency,  and  has  presented  the  formerly  quite 
unusual  feature  of  being  sometimes  the  precursor  of  gangrene 
of  the  lung.  Besides  these  two  definite  types  other  forms  also 
occur.  Thus  instead  of  a  fibrinous  material  the  exudation  may 

196 


TYPES   OF   PNEUMONIA  197 

be  of  a  serous,  haemorrhagic,  or  purulent  character.  Cases 
of  mixed  fibrinous  and  catarrhal  pneumonia  also  occur,  and 
in  the  catarrhal  there  may  be  great  leucocytic  emigration. 
Haemorrhages  also  may  occur  here. 

Besides  the  two  chief  types  of  pneumonia  there  is  another 
group  of  cases  which  are  somewhat  loosely  denominated  septic 
pneumonias,  and  which  may  arise  in  two  ways  :  (1)  by  the 
entrance  into  the  trachea  and  bronchi  of  discharges,  blood,  etc., 
which  form  a  nidus  for  the  growth  of  septic  organisms ;  these 
often  set  up  a  purulent  capillary  bronchitis  and  lead  to  infection 
of  the  air  cells  and  also  of  the  interstitial  tissue  of  the  lung ;  (2) 
from  secondary  pyogenic  infection  by  means  of  the  blood  stream 
from  suppurative  foci  in  other  parts  of  the  body.  (See  chapter 
on  Suppuration,  etc.)  In  these  septic  pneumonias  various 
changes,  resembling  those  found  in  the  other  types,  are  often 
seen  round  the  septic  foci. 

In  pneumonias,  therefore,  there  may  be  present  a  great  variety 
of  types  of  inflammatory  reaction.  We  shall  see  that  with  all  of 
them  bacteria  have  been  found  associated.  Special  importance 
is  attached  to  acute  croupous  pneumonia  on  account  of  its  course 
and  characters,  but  reference  will  also  be  made  to  the  other 
forms. 

Historical. — Acute  lobar  pneumonia  for  long  was  supposed  to  be  an 
effect  of  exposure  to  cold  ;  but  many  observers  were  dissatisfied  with 
this  view  of  its  etiology.  Not  only  did  cases  occur  where  no  such 
exposure  could  be  traced,  but  it  had  been  observed  that  the  disease 
sometimes  occurred  epidemically,  and  was  occasionally  contracted  by 
hospital  patients  lying  in  beds  adjacent  to  those  occupied  by  pneumonia 
cases.  Further,  the  sudden  onset  and  definite  course  of  the  disease  con- 
formed to  the  type  of  an  acute  infective  fever  ;  it  was  thus  suspected  by 
some  to  be  due  to  a  specific  infection.  This  view  of  its  etiology  was 
promulgated  in  1882-83  by  Friedlander,  whose  results  were  briefly  as 
follows.  In  pneumonic  lungs  there  were  cocci,  adherent  usually  in 
pairs,  and  possessed  of  a  definitely  contoured  capsule.  These  cocci 
could  be  isolated  and  grown  on  gelatin,  and  on  inoculation  in  mice  they 
produced  a  kind  of  septicaemia  with  inflammation  of  the  serous  membranes. 
The  blood  and  the  exudation  in  serous  cavities  contained  numerous 
capsulated  diplococci.  There  is  little  doubt  that  many  of  the  organisms 
seen  by  Friedlander  were  really  Fraenkel's  pneumococcus,  to  be  presently 
described. 

By  many  observers  it  had  been  found  that  the  sputum  of  healthy 
men,  when  injected  into  animals,  sometimes  caused  death,  with  the 
same  symptoms  as  in  the  case  of  the  injection  of  Friedlander's  coccus  ; 
and  in  the  blood  and  serous  exudations  of  such  animals  capsulated 
diplococci  were  found.  A.  Fraenkel  found  that  the  sputum  of  pneumonic 
patients  was  much  more  fatal  and  more  constant  in  its  effects  than  that 
of  healthy  individuals.  The  cocci  which  were  found  in  animals  dead  of 
this  "sputum  septicaemia,"  as  it  was  called,  differed  from  Friedlander's 
cocci  in  several  respects,  to  be  presently  studied.  Fraenkel  further 


198  THE   ACUTE   PNEUMONIAS 

investigated  a  few  cases  of  pneumonia,  and  isolated  from  them  cocci 
identical  in  microscopic  appearances,  cultures,  and  pathogenic  effects, 
with  those  isolated  in  sputum  septicaemia.  The  most  extensive  investi- 
gations on  the  whole  question  were  those  of  Weichselbaum,  published 
in  1886.  This  author  examined  129  cases  of  the  disease,  including 
cases  not  only  of  acute  croupous  pneumonia,  but  of  lobular  and  septic 
pneumonia.  From  them  he  isolated  four  groups  of  organisms.  (1) 
Diplococcus  pneumonice.  This  he  described  as  an  oval  or  lancet-formed 
coccus,  corresponding  in  appearance  and  growth  characters  to  Fraenkel's 
coccus.  (2)  Streptococcus  pneumonice.  This  on  the  whole  presented 
similar  characters  to  the  last,  but  it  was  more  vigorous  in  its  growth, 
and  could  grow  below  20°  C.,  though  it  preferred  a  temperature  of  37°  C. 
(3)  Staphylococcus  pyogencs  aureus.  (4)  JBacillus  pneumonice.  This  was 
a  rod-shaped  organism,  and  was  identical  with  Friedlander's  pneumo- 
coccus.  Of  these  organisms  the  diplococcus  pneumonias  was  by  far  the 
most  frequent.  It  also  occurred  in  all  forms  of  pneumonia.  Next  in 
frequency  was  the  streptococcus  pneumonias,  and  lastly  the  bacillus 
pneumonias.  Inoculation  experiments  were  also  performed  by  Weichsel- 
baum with  each  of  the  three  characteristic  cocci  he  isolated.  The 
diplococcus  pneumonias  and  the  streptococcus  pneumonias  both  gave 
pathogenic  effects  of  a  similar  kind  in  certain  animals. 

The  general  result  of  these  earlier  observations  was  to  establish 
the  occurrence  in  connection  with  pneumonia  of  two  species  of 
organisms,  each  having  its  distinctive  characters,  viz  : — 

1 .  Fraenkel 's  pneumococcus,  which  is  recognised  to  be  identical 
with  the  coccus  of  "  sputum  septicaemia,"  with  Weichselbaum 's 
diplococcus  pneumoniae,  and  with  his  streptococcus  pneumonia?. 

2.  Friedlander's  pneumococcus  (now  known  as  Friedlander's 
pneumobacillus),  which  is  almost  certainly  the  bacillus  pneu- 
monias of  Weichselbaum. 

We  shall  use  the  terms  "Fraenkel's  pneumococcus"  and 
"  Friedlander's  pneumobacillus,"  as  these  are  now  usually  applied 
to  the  two  organisms. 

Microscopic  Characters  of  the  Bacteria  of  Pneumonia.— 
Methods. — The  organisms  present  in  acute  pneumonia  can  best 
be  examined  in  film  preparations  made  from  pneumonic  lung 
(preferably  from  a  part  in  a  stage  of  acute  congestion  or  early 
hepatisation),  or  from  the  gelatinous  parts  of  pneumonic  sputum 
(here  again  preferably  when  such  sputum  is  either  rusty  or 
occurs  early  in  the  disease),  or  in  sections  of  pneumonic  lung. 
Such  preparations  may  be  stained  by  any  of  the  ordinary  weak 
stains,  such  as  a  watery  solution  of  methylene-blue,  but  Gram's 
method  is  to  be  preferred,  with  Bismarck-brown  or  Ziehl-Neelsen 
carbol-fuchsin  (one  part  to  ten  of  water)  as  a  contrast  stain ; 
with  the  latter  it  is  best  either  to  stain  for  only  a  few  seconds, 
or  to  overstain  and  then  decolorise  with  alcohol  till  the  ground 
of  the  preparation  is  just  tinted.  The  capsules  can  also  be 


BACTERIA   IN   PNEUMONIA  199 

stained  by  the  methods  already  described  (p.  102).  In  such 
preparations  as  the  above,  and  even  in  specimens  taken  from 
the  lungs  immediately  after  death  (as  may  be  quite  well  done 
by  means  of  a  hypodermic  syringe),  putrefactive  and  other 
bacteria  may  be  present,  but  those  to  be  looked  for  are  capsulated 
organisms,  which  may  be  of  either  or  both  of  the  varieties 
mentioned. 

(1)  Fraenkel's  Pneumococcus. — This  organism  occurs  in  the  form 
of  a  small  oval  coccus,  about  1  /^  in  longest  diameter,  arranged 
generally  in  pairs  (diplo- 

cocci),  but  also  in  chains  /" 

of  four  to  ten  (Fig.  64).  *",/;    * 

The  free  ends  are   often  .*,  3 

pointed  like  a  lancet,  hence  "  v  v>    * 

the  term  diplococcus  lance- 
olatus  has  also  been  ap-  *'/ 

plied   to  it.     These  cocci  ."_  „,• 

have  round  them  a  capsule,  .  ,-J; '' 

which,  in  films  stained  by 
ordinary  methods,  usually 
appears  as  an  unstained 
halo,  but  is  sometimes 
stained  more  deeply  than 
the  ground  of  the  pre- 
paration. This  difference 
in  staining  depends,  in  FIG.  64. — Film  preparation  of  pneumonic 
partat  least,  ontheamount  %utum>  Rowing  numerous  pneumococci 
L  £  ,  i  •  -•  i_«-'t  (Fraenkel  s)  with  unstained  capsules: 

of  decolorisation  to  which        ^ome  are  ar'ranged  in  short  chains>F 

the  preparation  has  been         Stained  with  carbol-fuchsiu.      x  1000. 
subjected.       The   capsule 

is  rather  broader  than  the  body  of  the  coccus,  and  has  a  sharply 
defined  external  margin.  This  organism  takes  up  the  basic 
aniline  stains  with  great  readiness,  and  also  retains  the  stain  in 
Gram's  method.  It  is  the  organism  of  by  far  the  most  frequent 
occurrence  in  true  croupous  pneumonia,  and  in  fact  may  be  said 
to  be  rarely  absent. 

(2)  Friedldnder1  s  Pneumobacillus. — As  seen  in  the   sputum 
and  tissues,  this  organism,  both  in  its  appearance  and  arrange- 
ment, as  also  in  the  presence  of  a  capsule,  somewhat  resembles 
Fraenkel's  pneumococcus,  and  it  was  at  first  described  as  the 
"  pneumococcus."     The  form,  however,  is  more  of  a  short  rod- 
shape,    and    it    has    blunt    rounded    ends ;    it   is   also   rather 
broader   than   Fraenkel's   pneumococcus.       It    is    now    classed 
amongst  the  bacilli,  especially  in  view  of  the  fact  that  elongated 


200 


THE   ACUTE   PNEUMONIAS 


rod  forms  may  occur  (Fig.  65).  The  capsule  lias  the  same 

general  characters  as 
that  of  FraenkePs  organ- 
ism.  Friedlander's 
pneumobacillus  stains 
readily  with  the  basic 
aniline  stains,  but  loses 
the  stain  in  Gram's 
method,  and  is  accord- 
ingly coloured  with  the 
contrast  stain, — fuchsin 
or  Bismarck -brown,  as 
above  recommended.  A 
valuable  means  is  thus 
afforded  of  distinguish- 
ing it  from  Fraenkel's 
pneumococcus  in  micro- 

FIG.  65. — Friedlander's  pneumobacillus,  showing    scopic  preparations. 


the  variations  in  length,  also  capsules.  Film 
preparation  from  exudate  in  a  case  of  pneit- 
monia.  x  1000. 


Friedlander's  organ- 
ism is  much  less  fre- 
quently present  in  pneu- 
is  associated  with  the 


inonia  than  Fraenkel's;    sometimes  it 
latter;    very    rarely    it 
occurs  alone. 

In  sputum  prepara- 
tions the  capsule  of  both 
pneumococci  may  not  be 
recognisable,  and  the 
same  is  sometimes  true 
of  lung  preparations. 
This  is  probably  due  to 
changes  which  occur  in 
the  capsule  as  the  result 
of  changes  in  the  vitality 
of  the  organisms.  Some- 
times in  preparations 
stained  by  ordinary 
methods  the  difficulty  of 
recognising  the  capsule 
when  it  is  present,  is 
due  to  the  refractive 
index  of  the  fluid  in 
which  the  specimen  is 
mounted  being  almost  identical  with  that  of  the  capsule. 


FIG.   66. — Fraenkel's  pneumococcus  in  serous 
exudation  at  site  of  inoculation  in  a  rabbit, 
showing  capsules  stained. 
Stained  by  Rd.  Muir's  method,      x  1000. 


This 


CULTIVATION   OF   PNEUMOCOCCUS 


201 


difficulty   can  always  be  overcome  by  having  the  groundwork 
of  the  preparation  tinted. 

The  Cultivation  of  Fraenkel's  Pneumococcus. — It  is  usually 
difficult,  and  sometimes  impossible,  to  isolate  this  coccus  directly 
from  pneumonic  sputum.  On  culture  media  it  has  not  a  vigorous 
growth,  and  when  mixed  with  other  bacteria  it  is  apt  to  be 
overgrown  by  the  latter.  To  get  a  pure  culture  it  is  best  to 
insert  a  small  piece  of  the  sputum  beneath  the  skin  of  a  rabbit 
or  a  mouse.  In  about  forty-eight  hours  the  animal  will  die, 
with  numerous  capsulated  pneumococci 
throughout  its  blood.  From  the  heart- 
blood  cultures  can  be  easily  obtained. 
Cultures  can  also  be  got  post  mortem  from 
the  lungs  of  pneumonic  patients  by 
streaking  a  number  of  agar  or  blood- 
agar  tubes  with  a  scraping  taken  from 
the  area  of  acute  congestion  or  commenc- 
ing red  hepatisation,  and  incubating  them 
at  37°  C.  The  colonies  of  the  pneumo- 
coccus  appear  as  almost  transparent  small 
discs  which  have  been  compared  to  drops 
of  dew  (Fig.  67).  This  method  is  also 
sometimes  successful  in  the  case  of 
sputum. 

The  appearances  presented  in  cultures  Fm.  67._Stroke  culture  of 
by  different  varieties  01  the  pneumococcus 
vary  somewhat.  It  always  grows  best 
on  blood  serum  or  on  Pfeiffer's  blood 
agar.  It  usually  grows  well  on  ordinary 
agar  or  in  bouillon,  but  not  so  well  on 
glycerin  agar.  In  a  stroke  culture  on 
blood  serum  growth  appears  as  an  almost  transparent  pellicle 
along  the  track,  with  isolated  colonies  at  the  margin.  On 
agar  media  it  is  more  manifest,  but  otherwise  has  similar 
characters.  The  appearances  are  similar  to  those  of  a  culture 
of  streptococcus  pyogenes,  but  the  growth  is  less  vigorous, 
and  is  more  delicate  in  appearance.  A  similar  statement  also 
applies  to  cultures  in  gelatin  at  22°  C.,  growth  in  a  stab  culture 
appearing  as  a  row  of  minute  points  which  remain  of  small 
size ;  there  is,  of  course,  no  liquefaction  of  the  medium.  On 
agar  plates  colonies  are  almost  invisible  to  the  naked  eye, 
but  under  a  low  power  of  the  microscope  appear  to  have  a 
compact  finely  granular  centre  and  a  pale  transparent  periphery. 
In  bouillon,  growth  forms  a  slight  turbidity,  which  settles  to  the 


Fraenkel's  pneumococcus 
on  blood  agar.  The 
colonies  are  large  and  un- 
usually distinct.  Twenty- 
four  hours'  growth  at 
37°  C.  Natural  size. 


202  THE   ACUTE   PNEUMONIAS 

bottom  of  the  vessel  as  a  slight  dust-like  deposit.  On  potatoes, 
as  a  rule,  no  growth  appears.  Cultures  on  such  media  may  be 
maintained  for  one  or  two  months,  if  fresh  sub-cultures  are  made 
every  four  or  five  days,  but  they  tend  ultimately  to  die  out. 
They  also  rapidly  lose  their  virulence,  so  that  four  or  five  days 
after  isolation  from  an  animal's  body  their  pathogenic  action 
is  already  diminished.  Eyre  and  Washbourn,  however,  have 
succeeded  in  maintaining  cultures  in  a  condition  of  constant 
virulence  for  at  least  three  months  by  growing  the  organisms  on 

agar  smeared  with  rab- 
bits' blood.      The   agar 

^  \         T  must  be  prepared  with 

J+     f***  r     **  Witte's    peptone,    must 

V  ^     s  no*  be  neated  over  100° 

*  i        \  C.,  and  after  neutralisa- 

tion (rosolic  acid  being 

v**  V*    |    >          —         used    as    the   indicator) 
^     must  have  '5  per  cent  of 
.^  ^  normal  sodium  hydrate 

\  %  v  added.     The  tubes  when 

s^  inoculated  are  to  be  kept 

•*        ^  \  at  37' 5°  C.  and  sealed  to 

prevent  evaporation.    In 
s»*  none  of  the  ordinary  arti- 

ficial media  do  pneumo- 
cocci  develop  a  capsule. 

ria.  DO. — rraenkel  s  pneuniococcus  from  a  pure    rp,  n 

culture  on  blood  agar  of  twenty-four  hours'    1.ney  usualJJ   appear   as 

growth,  some  iu  pairs,  some  in  short  chains,    diplococci,    but    in    pre- 

Stained  with  weak  carbol-fuchsin.     x  1000.       parations  made  from  the 

surface  of  agar  or  from 

bouillon,  shorter  or  longer  chains  may  be  observed  (Fig.  68). 
After  a  few  days'  growth  they  lose  their  regular  shape  and  size, 
and  involution  forms  appear.  Usually  the  pneumococcus  does 
not  grow  below  22°  C.,  but  forms  in  which  the  virulence  has 
disappeared  often  grow  well  at  20°  C.  Its  optimum  temperature 
is  37°  C.,  its  maximum  42°  C.  It  is  preferably  an  aerobe,  but 
can  exist  without  oxygen.  It  prefers  a  slightly  alkaline  medium 
to  a  neutral,  and  does  not  grow  on  an  acid  medium.  These 
facts  show  that  when  growing  outside  the  body  on  artificial 
media,  the  pneumococcus  is  a  comparatively  delicate  organism. 
There  has  been  described  by  Eyre  and  Washbourn  a  non- 
pathogenic  type  of  the  pneumococcus  which  may  be  found  in 
the  healthy  mouth,  and  which  may  also  be  produced  during  the 
saprophytic  growth  of  the  virulent  form.  From  the  latter  it 


CULTIVATION   OF    PNEUMOBACILLUS 


203 


differs  generally  in  its  more  vigorous  growth,  in  producing  a 

uniform  cloud  in  bouillon,  in  slowly  liquefying  gelatin,  and  in 

growing  on  potato. 

The   Cultivation  of  Friedlander's    Pneumobacillus.— This 

organism,  when  present  in  sputum  or  in  a  pneumonic  lung,  can 
be  readily  separated  by  making  ordinary 
gelatin  plate  cultures,  or  a  series  of  successive 
strokes  on  agar  tubes.  The  surface  colonies 
always  appear  as  white  discs  which  become 
raised  from  the  surface  so  as  to  appear  like 
little  knobs  of  ivory.  From  these,  pure 
cultures  can  be  readily  obtained.  The  ap- 
pearance of  a  stab  culture  in  gelatin  growth 
is  very  characteristic.  At  the  site  of  the 
puncture,  there  is  on  the  surface  a  white 
growth  heaped  up,  it  may  be  fully  one-eighth 
of  an  inch  above  the  level  of  the  gelatin ; 
along  the  needle  track  there  is  a  white 


FIG.  69. — Stab  culture 
of  Friedlander's 
pneumobacillus  in 
peptone  gelatin, 
showing  the  riail- 
like  appearance  ; 
ten  days'  growth. 
Natural  size. 


FIG.     70.  —  Friedlander's     pneumobacillus,1 
from  a   young  culture  on  agar,  showing 
some  rod-shaped  forms. 
Stained  with  thioniu-blue.      x  1000. 


granular  appearance,  so  that  the  whole  resembles  a  white  round- 
headed  nail  driven  into  the  gelatin  (Fig.  69).     Hence  the  name 

1  The  apparent  size  of  this  organism,  on  account  of  the  nature  of  its  sheath, 
varies  much  according  to  the  stain  used.  If  stained  with  a  strong  stain,  e.g. 
carbol-fuchsin,  its  thickness  appears  nearly  twice  as  great  as  is  shown  in  the 


204  THE   ACUTE   PNEUMONIAS 

"nail-like"  which  has  been  applied.  Occasionally  bubbles  of 
gas  develop  along  the  line  of  growth.  There  is  no  liquefaction 
of  the  medium.  On  sloped  agar  it  forms  a  very  white  growth 
with  a  shiny  lustre,  which,  when  touched  with  a  platinum  needle, 
is  found  to  be  of  a  viscous  consistence.  In  cultures  much  longer 
rods  are  formed  than  in  the  tissues  of  the  body  (Fig.  70).  On 
the  surface  of  potatoes  it  forms  an  abundant  moist  white  layer. 
Friedlander's  bacillus  has  active  fermenting  powers  on  sugars, 
though  varieties  isolated  by  different  observers  vary  in  the  degree 
in  which  such  powers  are  possessed.  It  always  seems  capable 
of  acting  on  dextrose,  lactose,  maltose,  dextrin,  and  mannite,  and 
sometimes  also  on  glycerin.  The  substances  produced  by  the 
fermentation  vary  with  the  sugar  fermented,  but  include  ethylic 
alcohol,  acetic  acid,  laevolactic  acid,  succinic  acid,  along  with 
hydrogen  and  carbonic  acid  gas.  The  amount  of  acid  produced 
from  lactose  seems  only  exceptionally  sufficient  to  cause  coagula- 
tion of  milk.  It  is  said  by  some  that  this  bacillus  is  identical 
with  an  organism  common  in  sour  milk,  and  also  a  normal 
inhabitant  of  the  human  intestine,  viz.  the  bacterium  lactis 
serogenes  of  Escherich. 

The  Occurrence  of  the  Pneumobacteria  in  Pneumonia  and 
other  Conditions. — Capsulated  organisms  have  been  found  in 
every  variety  of  the  disease — in  acute  croupous  pneumonia,  in 
broncho-pneumonia,  in  septic  pneumonia.  In  the  great  majority 
of  these  it  is  Fraenkel's  pneumococcus  which  both  microscopic- 
ally and  culturally  has  been  found  to  be  present.  Friedlander's 
pneumobacillus  occurs  in  only  about  5  per  cent  of  the  cases.  It 
may  be  present  alone  or  associated  with  Fraenkel's  organism.  In 
a  case  of  croupous  pneumonia  the  pneumococci  are  found  all 
through  the  affected  area  in  the  lung,  especially  in  the  exudation 
in  the  air-cells.  They  also  occur  in  the  pleural  exudation  and 
effusion,  and  in  the  lymphatics  of  the  lung.  The  greatest  number 
are  found  in  the  parts  where  the  inflammatory  process  is  most 
recent,  e.g.  in  an  area  of  acute  congestion  in  a  case  of  croupous 
pneumonia,  and  therefore  such  parts  are  preferably  to  be  selected 
for  microscopic  examination,  and  as  the  source  of  cultures.  Some- 
times there  occur  in  pneumonic  consolidation  areas  of  suppura- 
tive  softening,  which  may  spread  diffusely.  In  such  areas  the 
pneumococci  occur  with  or  without  ordinary  pyogenic  organisms, 
streptococci  being  the  commonest  concomitants.  In  other  cases, 
especially  when  the  condition  is  secondary  to  influenza,  gangrene 
may  supervene  and  lead  to  destruction  of  large  portions  of  the 
lung.  In  these  a  great  variety  of  bacteria,  both  aerobes  and 
anaerobes,  are  to  be  found. 


DISTRIBUTION    OF   PNEUMOBACTERIA        205 

In  ordinary  broncho -pneumonias  also  Fraenkel's  pneumo- 
coccus  is  usually  present,  sometimes  along  with  pyogenic  cocci ; 
in  the  broncho-pneumonias  secondary  to  diphtheria  it  may  be 
accompanied  by  the  diphtheria  bacillus,  and  also  by  pyogenic 
cocci ;  in  typhoid  pneumonias  the  typhoid  bacillus  or  the  b.  coli 
may  be  alone  present  or  be  accompanied  by  the  pneumococcus, 
and  in  influenza  pneumonias  the  influenza  bacillus  may  occur. 
In  septic  pneumonias  the  pyogenic  cocci  in  many  cases  are  the 
only  organisms  discoverable,  but  the  pneumococcus  may  also  be 
present.  Especially  important,  as  we  shall  see,  from  the  point 
of  view  of  the  etiology  of  the  disease,  is  the  occurrence  in  other 
parts  of  the  body  of  pathological  conditions  associated  with  the 
presence  of  the  pneumococcus.  By  direct  extension  to  neigh- 
bouring parts  empyema,  pericarditis,  and  lymphatic  enlargements 
in  the  mediastinum  and  neck  may  take  place ;  in  the  first  the 
pneumococcus  may  occur  either  alone  or  with  pyogenic  cocci. 
But  distant  parts  may  be  affected,  and  the  pneumococcus  may  be 
found  in  suppurations  and  inflammations  in  various  parts  of  the 
body  (subcutaneous  tissue,  peritoneum,  joints,  kidneys,  liver,  etc.), 
in  otitis  media,  ulcerative  endocarditis  (p.  188),  and  meningitis. 
These  conditions  may  take  place  either  as  complications  of 
pneumonia,  or  they  may  constitute  the  primary  disease.  The 
occurrence  of  meningitis  is  of  special  importance,  for  next  to  the 
lungs  the  meninges  appear  to  be  the  parts  most  liable  to  attack 
by  the  pneumococcus.  A  large  number  of  cases  have  been 
investigated  by  Netter,  who  gives  the  following  tables  of  the 
relative  frequency  of  the  primary  infections  by  the  pneumococcus 
in  man  : — 

(1)  In  adults- 
Pneumonia        .         ......         65 '95  per  cent 

Broncho-pneumonia)  -IK  QK 

Capillary  bronchitis/        •     -    •         • 
Meningitis  .  -.  13 '00 


Empyema 
Otitis 

Endocarditis 
Liver  abscess 


8-53 
2-44 
1-22 
1-22 


(2)  In  children  46  cases  were  investigated.  In  29  the  primary  affection 
was  otitis  media,  in  12  broncho-pneumonia,  in  2  meningitis,  in  1 
pneumonia,  in  1  pleurisy,  in  1  pericarditis. 

Thus  in  children  the  primary  source  of  infection  is  in  a  great 
many  cases  an  otitis  media,  and  Netter  concludes  that  infection 
takes  place  in  such  conditions  from  the  nasal  cavities. 

As   bearing  on    the    occurrence  of   pneumococcal  infections 


206 


THE   ACUTE   PNEUMONIAS 


secondary  to  such  a  local  lesion  as  pneumonia,  it  is  important  to 
note  that  in  a  large  proportion  of  cases  of  the  latter  disease  the 
pneumococcus  can  be  isolated  from  the  blood. 

Experimental  Inoculation. — The  pneumococcus  of  Fraenkel  is 
pathogenic  to  various  animals,  though  the  effects  vary  somewhat 
with  the  virulence  of  the  race  used.  The  susceptibility  of 
different  species,  as  Gamaleia  has  shown,  varies  to  a  considerable 


FIG.  71. — Capsulated  pneumococci  in  blood  taken  from  the  heart  of  a 
rabbit,  dead  after  inoculation  with  pneumonic  sputum. 

Dried  film,  fixed  with  corrosive  sublimate.  Stained  with  carbol-fuchsin  and 
partly  decolorised.  x  1000. 

extent.  The  rabbit,  and  especially  the  mouse,  are  very  sus- 
ceptible ;  the  guinea-pig,  the  rat,  the  dog,  and  the  sheep 
occupy  an  intermediate  position  ;  the  pigeon  is  immune.  In 
the  more  susceptible  animals  the  general  type  of  the  disease 
produced  is  not  pneumonia,  but  a  general  septicaemia.  Thus,  if 
a  rabbit  or  a  mouse  be  injected  subcutaueously  with  pneumonic 
sputum,  or  with  a  scraping  from  a  pneumonic  lung,  death 
occurs  in  from  twenty-four  to  forty-eight  hours.  There  is  some 
fibrinous  infiltration  at  the  point  of  inoculation,  the  spleen  is 


EXPERIMENTAL   INOCULATION  207 

often  enlarged  and  firm,  and  the  blood  contains  capsulated 
pneumococci  in  large  numbers  (Fig.  71).  If  the  seat  of  inocula- 
tion be  in  the  lung,  there  generally  results  pleuritic  effusion  on 
both  sides,  and  in  the  lung  there  may  be  a*  process  somewhat 
resembling  the  early  stage  of  acute  croupous  pneumonia  in  man. 
There  are  often  also  pericarditis  and  enlargement  of  spleen. 
We  have  already  stated  that  cultures  of  the  pneumococci  on 
artificial  media  in  a  few  days  begin  to  lose  their  virulence. 
Now,  if  such  a  partly  attenuated  culture  be  injected  sub- 
cutaneously  into  a  rabbit,  there  is  greater  local  reaction  ; 
pneumonia,  with  exudation  of  lymph  on  the  surface  of  the 
pleura,  and  a  similar  condition  in  the  peritoneum,  may  occur. 
In  sheep  greater  immunity  is  marked  by  the  occurrence,  after 
subcutaneous  inoculation,  of  an  enormous  local  sero-fibrinous 
exudation,  and  by  the  fact  that  few  pneumococci  are  found  in 
the  blood  stream.  In tra- pulmonary  injection  in  sheep  is 
followed  by  a  typical  pneumonia,  which  is  generally  fatal.  The 
dog  is  still  more  immune  •  in  it  also  intra-pulmonary  injection  is 
followed  by  a  fibrinous  pneumonia,  which  is  only  sometimes 
fatal.  Inoculation  by  inhalation  appears  only  to  have  been 
performed  in  the  susceptible  mouse  and  rabbit ;  here  also 
septicaemia  resulted. 

The  general  conclusion  to  be  drawn  from  these  experiments 
thus  is  that  in  highly  susceptible  animals  virulent  pneumococci 
produce  a  general  septicasmia ;  whereas  in  more  immune  species 
there  is  an  acute  local  reaction  at  the  point  of  inoculation,  and 
if  the  latter  be  in  the  lung,  then  there  may  result  pneumonia, 
which,  of  course,  is  merely  a  local  acute  inflammation  occurring 
in  a  special  tissue,  but  identical  in  essential  pathology  with  an 
inflammatory  reaction  in  any  other  part  of  the  body.  When  a 
dose  of  pneumococci  sufficient  to  kill  a  rabbit  is  injected  sub- 
cutaneously  in  the  human  subject,  it  gives  rise  to  a  local  inflam- 
matory swelling  with  redness  and  slight  rise  of  temperature,  all 
of  which  pass  off  in  a  few  days.  It  is  therefore  justifiable  to 
suppose  that  man  occupies  an  intermediate  place  in  the  scale  of 
susceptibility,  probably  between  the  dog  and  the  sheep,  and 
that  when  the  pneumococcus  gains  an  entrance  to  his  lungs,  the 
local  reaction  in  the  form  of  pneumonia  occurs.  In  this  con- 
nection the  occurrence  of .  manifestations  of  general  infection 
associated  with  pneumonia  in  man  is  of  the  highest  import- 
ance. We  have  seen  that  meningitis  and  other  inflammations 
are  not  very  rare  complications  of  the  disease,  and  such  cases 
form  a  link  connecting  the  local  disease  in  the  human  subject 
with  the  general  septicaBmic  processes  which  may  be  produced 


208  THE   ACUTE   PNEUMONIAS 

artificially  in  the  more  susceptible  representatives  of  the  lower 
animals. 

A  fact  which  at  first  appeared  rather  to  militate  against  the 
pneumococcus  beirtg  the  cause  of  pneumonia  was  the  discovery 
of  this  organism  in  the  saliva  of  healthy  men.  This  fact  was 
early  pointed  out  by  Pasteur,  and  also  by  Fraenkel,  and  the 
observation  has  been  confirmed  by  many  other  observers.  It 
can  certainly  be  isolated  from  the  mouths  of  a  considerable 
proportion  of  normal  men,  from  their  nasal  cavities,  etc.,  being 
probably  in  any  particular  individual  more  numerous  at  some 
times  than  at  others,  and  sometimes  being  entirely  absent. 
This  can  be  proved,  of  course,  by  inoculation  of  susceptible 
animals.  Such  a  fact,  however,  only  indicates  the  importance 
of  predisposing  causes  in  the  etiology  of  the  disease,  and  it  is 
further  to  be  observed  that  we  have  corresponding  facts  in  the 
case  of  the  diseases  caused  by  pyogenic  staphylococci,  streptococci, 
the  bacillus  coli,  etc.  It  is  probable  that  by  various  causes  the 
vitality  and  power  of  resistance  of  the  lung  are  diminished,  and 
that  then  the  pneumococcus  gains  an  entrance.  In  relation  to 
this  possibility  we  have  the  very  striking  facts  that  in  the 
irregular  forms  of  pneumonia,  secondary  to  such  conditions  as 
typhoid  and  diphtheria,  the  pneumococcus  is  very  frequently 
present,  alone  or  with  other  organisms.  Apparently  the  effects 
produced  by  such  bacteria  as  the  b.  typhosus  and  the  b. 
diphtherise  can  devitalise  the  lung  to  such  an  extent  that 
secondary  infection  by  the  pneumococcus  is  more  likely  to  occur 
and  set  up  pneumonia.  We  can  therefore  understand  how 
much  less  definite  devitalising  agents  such  as  cold,  alcoholic 
excess,  etc.,  can  play  an  important  part  in  the  causation  of 
pneumonia.  In  this  way  also  other  abnormal  conditions  of  the 
respiratory  tract,  a  slight  bronchitis,  etc.,  may  play  a  similar 
part. 

It  is  more  difficult  to  explain  why  sometimes  the  pneumo- 
coccus is  associated  with  a  spreading  inflammation,  as  in  croupous 
pneumonia,  whilst  at  other  times  it  is  localised  to  the  catarrhal 
patches  in  broncho-pneumonia.  It  is  quite  likely  that  in  the 
former  condition  the  organism  is  possessed  of  a  different  order 
of  virulence,  though  of  this  we  have  no  direct  proof.  We  have, 
however,  a  closely  analogous  fact  in  the  case  of  erysipelas ;  this 
disease,  we  have  stated  reasons  for  believing,  is  produced  by  a 
streptococcus  which,  when  less  virulent,  causes  only  local 
inflammatory  and  suppurative  conditions. 

Summary. — We  may  accordingly  summarise  the  facts  re- 
garding the  relation  of  Fraenkel's  pneumococcus  to  the  disease 


PNEUMOCOCCUS   INFECTION  209 

by  saying  that  it  can  be  isolated  from  nearly  all  cases  of  acute 
croupous  pneumonia,  and  also  from  a  considerable  proportion 
of  other  forms  of  pneumonia.  When  injected  into  the  lungs  of 
moderately  insusceptible  animals  it  gives  rise  to  pneumonia.  If, 
in  default  of  the  crucial  experiment  of  intra-pulmonary  injection 
in  the  human  subject,  we  take  into  account  the  facts  we  have 
discussed,  we  are  justified  in  holding  that  it  is  the  chief  factor  in 
causing  croupous  pneumonia,  and  also  plays  an  important  part 
in  other  forms.  Pneumonia,  in  the  widest  sense  of  the  term,  is, 
however,  not  a  specific  affection,  and  various  inflammatory  con- 
ditions in  the  lungs  can  be  set  up  by  the  different  pyogenic 
organisms,  by  the  bacilli  of  diphtheria,  of  influenza,  etc. 

The  possibility  of  Friedlander's  pneumobacillus  having  an 
etiological  relationship  to  pneumonia  has  been  much  disputed. 
Its  discoverer  found  that  it  was  pathogenic  towards  mice  and 
guinea-pigs,  and  to  a  less  extent  towards  dogs.  Rabbits  appeared 
to  be  immune.  The  type  of  the  disease  was  of  the  nature  of  a 
septicaemia.  No  extended  experiments,  such  as  those  performed 
by  Gamaleia  with  FraenkePs  coccus,  have  been  done,  and  there- 
fore we  cannot  say  whether  any  similar  pneumonic  effects  are 
produced  by  it  in  partly  susceptible  animals.  The  organism 
appears  to  be  present  alone  in  a  small  number  of  cases  of 
pneumonia,  and  the  fact  that  it  also  appears  to  have  been  the 
only  organism  present  in  certain  septicsemic  complications  of 
pneumonia,  such  as  empyema  and  meningitis,  render  it  possible 
that  it  may  be  the  causal  agent  in  a  few  cases  of  the  disease. 

In  the  septic  pneumonias  the  different  pyogenic  organisms 
already  described  are  found,  and  sometimes  in  ordinary 
pneumonias,  especially  the  catarrhal  forms,  other  organisms, 
such  as  the  b.  coli  or  its  allies,  may  be  the  causal  agents. 

The  Pathology  of  Pneumococcus  Infection. — The  effects  of 
the  action  of  the  pneumococcus,  at  any  rate  in  a  relatively 
insusceptible  animal  such  as  man,  seem  to  indicate  that  toxins 
must  play  an  important  part.  Pneumonia  is  a  disease  which 
presents  in  many  respects  the  characters  of  an  acute  poisoning. 
In  very  few  cases  does  death  take  place  from  the  functions  of 
the  lungs  being  interfered  with  to  such  an  extent  as  to  cause 
asphyxia.  It  is  from  cardiac  failure,  from  grave  interference 
with  the  heat-regulating  mechanism,  and  from  general  nervous 
depression  that  death  usually  results.  These  considerations, 
taken  in  connection  with  the  fact  that  in  man  the  organisms  are 
found  in  the  greatest  numbers  in  the  lung,  suggest  that  a  toxic 
action  is  at  work.  Various  attempts  have  been  made  to  isolate 
toxins  from  cultures  of  the  pneumococcus,  e.g.  by  precipitating 
14 


210  THE   ACUTE   PNEUMONIAS 

bouillon  cultures  with  alcohol  or  ammonium  sulphate,  and 
poisonous  effects  have  been  produced  by  certain  substances  thus 
derived ;  but  the  effects  produced  are,  as  in  so  many  other 
similar  cases,  of  a  non-specific  character  and  to  be  classed  as 
interferences  with  general  metabolism.  The  general  conclusion 
has  been  that  the  toxins  at  work  in  pneumonia  are  intracellular  ; 
but  no  special  light  has  been  thrown  on  the  common  effects  of 
the  members  of  this  group  of  bacterial  poisons. 

Immunisation  against  the  Pneumococcus. — Animals  can  be 
immunised  against  the  pneumococcus  by  inoculation  with 
cultures  which  have  become  attenuated  by  growth  on  artificial 
media,  or  with  the  naturally  attenuated  cocci  which  occur  in  the 
sputum  after  the  crisis  of  the  disease.  Netter  effected  immun- 
isation by  injecting  an  emulsion  of  the  dried  spleen  of  an  animal 
dead  of  pneumococcus  septicaemia.  Virulent  cultures  killed  by 
heating  at  62°  C.,  rusty  sputum  kept  at  60°  C,  for  one  to  two 
hours  and  then  filtered,  and  filtered  or  unfiltered  bouillon 
cultures  similarly  treated  have  also  been  used.  In  all  cases  one 
or  two  injections,  at  intervals  of  several  days,  are  sufficient  for 
immunisation,  but  the  immunity  has  often  been  observed  to 
be  of  a  fleeting  character  and  may  not  last  more  than  a  few 
weeks.  The  serum  of  such  immunised  animals  protects  rabbits 
against  subsequent  inoculation  with  pneumococci,  and  if  injected 
within  twenty-four  hours  after  inoculation,  prevents  death.  A 
protective  serum  was  obtained  by  Washbourn,  who  employed 
pneumococcus  cultures  of  constant  virulence.  This  observer 
immunised  a  pony  by  using  successively  (1)  broth  cultures  killed 
by  one  hour's  exposure  to  60°  C. ;  (2)  living  agar  cultures ;  (3) 
living  broth  cultures.  From  this  animal  there  was  obtained  a 
serum  which  protected  susceptible  animals  against  many  times 
an  otherwise  fatal  dose,  and  which  also  had  a  limited  curative 
action.  It  is  stated  that  the  serum  of  patients  who  have 
recovered  from  pneumonia  has  in  a  certain  proportion  of  cases 
a  protective  effect  against  the  pneumococcus  in  rabbits,  similar 
to  that  exhibited  by  the  serum  of  immune,  animals. 

The  Klemperers  treated  a  certain  number  of  cases  of  human 
pneumonia  by  serum  derived  from  immune  animals,  and  appar- 
ently with  a  certain  measure  of  success,  and  sera  prepared  by 
Washbourn  and  by  others  have  also  been  used.  The .  results 
obtained  by  different  observers  have,  however,  been  rather  con- 
tradictory. The  use  of  these  sera  apparently  causes  the  tempera- 
ture in  some  cases  to  fall,  and  even  may  hasten  a  crisis,  but 
further  experience  is  necessary  before  their  value  in  therapeutics 
can  be  properly  estimated. 


PNEUMOCOCCUS   INFECTION  211 

There  has  been  considerable  difference  of  opinion  as  to  the 
explanations  to  be  given  of  the  facts  observed  regarding 
immunisation  against  the  pneumococcus  and  especially  regarding 
the  properties  of  immune  sera.  At  first  these  sera  were  supposed 
to  possess  antitoxic  qualities — largely  on  the  ground  that  no 
bactericidal  effect  was  produced  by  them  on  the  bacterium  in 
vitro.  As  no  specific  toxin  has  been  proved  to  be  concerned  in 
the  action  of  the  organism  the  development  of  an  antitoxin 
during  immunisation  must,  in  the  present  state  of  knowledge, 
be  looked  on  as  not  yet  proved.  To  explain  the  action  of  a 
serum  in  preventing  and  curing  pneumococcal  infections,  it 
has  been  thought  to  have  the  complex  character  seen  in  anti- 
typhoid sera  in  which  two  substances — immune  body  and  com- 
plement (see  Immunity) — are  concerned,  and  the  variability  in 
the  therapeutic  results  obtained  has  been  accounted  for  on 
the  view  that  there  might  be  a  deficiency  of  complement,  such 
as  occurs  in  other  similar  cases.  The  absence  of  bactericidal 
effect,  however,  raises  several  difficult  points.  It  is  stated  that 
no  such  effect  is  observable  either  in  immune  sera,  or  in  the 
serum  of  patients  who  have  successfully  come  through  an  attack 
of  the  natural  disease.  Some  effect  of  the  kind  would  be  ex- 
pected to  be  present  if  the  anti-pneumonic  serum  were  quite 
comparable  to  the  antityphoid  serum.  Within  recent  times 
many  have  turned  to  the  opsonic  property  of  sera  to  account 
for  the  facts  observed.  In  this  connection  Mennes  observed 
that  normal  leucocytes  only  become  phagocytic  towards  pneumo- 
cocci  when  they  are  lying  in  the  serum  of  an  animal  immunised 
against  this  bacterium.  Wright  had  in  his  early  papers  looked  to 
the  phagocytosis  of  sensitised  bacteria  to  explain  their  destruction 
in  the  absence  of  bactericidal  qualities  in  the  serum  alone,  and 
Neufeld  and  Bimpau  have  described  the  occurrence  of  an  opsonic 
effect  in  the  action  of  an  anti-pneumococcic  serum.  Further 
work  may  show  that  along  these  lines  lies  the  explanation  of  the 
facts  observed. 

In  studying  further  the  relationship  of  the  opsonic  effect  to 
pneumococcal  infection,  inquiry  has  been  directed  to  the  opsonic 
qualities  of  the  blood  of  pneumonic  patients,  especially  with  a 
view  to  throwing  light  on  the  nature  of  the  febrile  crisis. 
According  to  some  results  the  opsonic  index  as  compared  with 
that  of  a  healthy  person  is  not  above  normal,  but  if  the  possible 
phagocytic  capacities  of  the  whole  blood  of  the  sick  person  be 
taken  into  account  these  will  probably  be  much  above  normal  in 
consequence  of  the  leucocytosis  which  usually  accompanies  a 
successful  resistance  to  this  infection.  It  has  been  observed, 


212  THE   ACUTE   PNEUMONIAS 

however,  that  as  the  crisis  approaches  in  a  case  which  is  to 
recover  the  opsonic  index  rises,  and  after  defervescence  gradually 
falls  to  normal.  And  further,  as  bearing  on  the  factors  in- 
volved in  the  successful  resistance  of  the  organism  to  the 
pneumococcus,  it  has  been  noted  that  avirulent  pneumococci  are 
more  readily  opsonised  than  more  virulent  strains.  Further 
observations  along  such  lines  are  to  be  looked  for  with  interest, 
and  it  may  be  said  that  Wright's  vaccination  methods  have  been 
applied  to  the  treatment  of  pneumonia  cases,  and  in  certain 
instances  are  said  to  have  been  followed  by  favourable  result. 
It  may  be  noted  here,  in  conclusion,  that  in  man  it  is  probable 
that  immunity  against  pneumonia  may  be  short-lived,  as  in  a 
good  many  cases  of  pneumonia  a  history  of  a  previous  attack  is 
elicited. 

Agglutination  of  the  Pneumococcus. — If  a  small  amount  of  a 
culture  of  Fraenkel's  pneumococcus  be  placed  in  an  anti-pneumo- 
coccic  serum,  an  aggregation  of  the  bacteria  into  clumps  occurs. 
Such  an  agglutination,  as  it  is  called,  is  frequently  observed 
under  similar  circumstances  with  other  bacteria.  The  pheno- 
menon is  not  invariably  associated  with  the  presence  of  protective 
bodies  in  a  serum,  but  it  has  been  used  for  diagnostic  purposes 
in  the  differentiation  of  sore  throats  due  to  pneumococcus 
infection  from  those  due  to  other  bacteria.  Whether  the  method 
is  reliable  has  still  to  be  proved. 

Methods  of  Examination. —  These  have  been  already 
described,  but  may  be  summarised  thus :  (1)  Microscopic. 
Stain  films  from  the  densest  part  of  the  sputum  or  from 
the  area  of  spreading  inflammation  in  the  lung  by  Gram's 
method  and  by  carbol-fuchsin,  etc.  (pp.  99,  101),  in  the  latter 
case  without  decolorising  the  groundwork  of  the  preparation. 

(2)  By  cultures,  (a)  FraenkeVs  pneumococcus.  With  similar 
material  make  successive  strokes  on  agar,  blood  agar,  or  blood 
serum.  The  most  certain  method,  however,  is  to  inject  some 
of  the  material  containing  the  suspected  cocci  into  a  rabbit.  If 
the  pneumococcus  be  present  the  animal  will  die,  usually  within 
forty-eight  hours,  with  numerous  capsulated  pneumococci  in  its 
heart  blood.  With  the  latter  inoculate  tubes  of  the  above  media 
and  observe  the  growth.  In  some  cases  of  severe  pneumococcic 
infection  the  organism  may  be  cultivated  from  the  blood  obtained 
by  venesection  (p.  68).  (6)  Friedldnder 's  pneumobacillus  can 
be  readily  isolated  either  by  ordinary  gelatin  plates  or  by 
successive  strokes  on  agar  media. 


EPIDEMIC   CEREBRO-SPINAL   MENINGITIS     213 


EPIDEMIC  CEREBKO-SPINAL  MENINGITIS. 

As  the  result  of  observations  on  this  disease  in  different  parts 
of  the  world,  it  has  been  now  established  that  the  causal  agent 
is  the  diplococcus  intracellularis  meningitidis  first  described  by 
Weichselbaum.  This  organism  is  a  small  coccus  measuring 
about  1  ft  in  diameter  and  usually  occurs  in  pairs,  the  adjacent 
sides  being  somewhat  flattened  against  each  other.  In  most 
cases  the  cocci  are  chiefly  contained  within  polymorphonuclear 
leucocytes  in  the  exudation  (Fig.  72) ;  in  some  cases,  however, 
the  majority  may  be 
lying  free.  It  stains 
readily  with  basic  aniline 
dyes,  but  loses  the  stain 
in  Gram's  method,  the 
readiness  with  which  the 
organism  decolorises 
varying  with  different 
strains.  Both  in  appear- 
ance and  in  its  staining 
reactions  it  is  superfici- 
ally similar  to  the  gono- 
coccus  (vide  infra).  The 
organism  can  readily  be 
cultivated  outside  the 
body,  but  the  conditions 
of  growth  are  somewhat 
restricted — agar  with  an 
admixture  of  serum  or 
blood  (preferably  human) 
is  most  suitable.1  Strains 

separated  in  different  epidemics  appear  to  present  slight  in- 
dividual variations,  but  the  following  description  may  be  taken 
as  summing  up  the  common  characters.  Growth  takes  place 
best  at  the  temperature  of  the  body,  and  practically  ceases 
at  25°  C.  On  serum  agar  the  colonies  are  circular  discs  of 
almost  transparent  appearance  and  possessing  a  smooth  shining 
surface ;  they  have  little  tendency  to  become  confluent.  When 
examined  under  a  low.  magnification  the  colour  is  seen  to  be 
somewhat  yellowish  and  the  margins  usually  are  smooth  and 

1  A  very  good  medium  is  one  composed  of  1  part  of  ascitic  fluid  and  6 
parts  of  1  per  cent  glucose  agar  ;  the  serum  obtained  aseptically  is  added  to 
the  agar  in  the  melted  state  at  45°  C.  and  the  tubes  are  tested  as  regards 
sterility  by  incubation. 


FIG.  72. — Film  preparation  of  exudation  from 
a  case  of  meningitis,  showing  the  diplococci 
within  leucocytes. 
Stained  with  carbol-thionin-blue.      x  1000. 


214  THE   ACUTE   PNEUMONIAS 

regular,  though  on  some  media  slight  crenation  may  appear.  The 
colonies  may  be  of  considerable  size,  reaching  sometimes  a 
diameter  of  2  to  3  mm.  on  the  third  day.  On  plain  agar  the 
colonies  are  very  much  smaller,  and  sometimes  no  growth 
occurs  ;  sub-cultures  especially  often  fail  to  give  any  growth 
on  this  medium.  In  serum  bouillon  the  organism  produces  a 
general  turbidity  with  formation  of  some  deposit  after  a  day  or 
two.  It  ferments  maltose,  galactose,  and  dextrose  with  acid 
production,  a  property  which  distinguishes  it  from  the  micro- 
coccus  catarrhalis  (vide  infra).  Buchanan  has  pointed  out  that 
this  may  be  demonstrated  by  making  up  lots  of  Loffler's  medium 

(p.  40)    in  Petri   dishes 
•  with  each  of  these  sugars 

"  *    •*   0  added.       In     all     cases 

£         *."*.,*         S  »  •-  growth  occurs  best  when 

•'  *    „***  ;•  .  the  medium  has  a  neutral 

»       -  v*    ^*  #    *»   *        %!jffc       or  very  slightly  alkaline 

^  ^  *     -*,  ««  **.*  *%     *.t      »  **4»  *      reaction.    In  cultures  the 
>f        »»*  "**«.*    *         *"^t"V  •-     •*    «     organism    presents     the 
•  *%****  '•  *••       *     same  appearance  as  in  the 

****'      «•          9       «      ,    "•  •   body   and    often    shows 
*  **  tetrad  formation.     There 

is  also  a  great  tendency 


% 
*'      *"** 


,  production  of  in- 

^  ^                     *.     t      ..  '  volution  forms  (Fig.  73), 

»jfcn»  ^  ^  many  of    the    cocci    be- 

%  coming     much    swollen, 

FIG.  73,-Pure  culture  of  diplococcus  intra-     staining  ^^  a-nd  aftei>- 
cellularis,  showing  involution  forms.  wards  undergoing  disin- 

tegration.    This  change, 

according  to  Flexner's  observations,  would  appear  to  b<3  due  to 
the  production  of  an  autolytic  enzyne,  and  he  has  also  found  that 
this  substance  has  the  property  of  producing  dissolution  of  the 
bodies  of  other  bacteria.  The  life  of  the  organism  in  cultures  is 
a  comparatively  short  one  ;  after  a  few  days  cultures  will  often 
be  found  to  be  dead,  but,  by  making  sub-cultures  every  three  or 
four  days  strains  can  be  maintained  alive  for  considerable  periods. 
The  organism  is  readily  killed  by  heat  at  60°  C.,  and  it  is  also 
very  sensitive  to  weak  antiseptics  ;  drying  for  a  period  of  a  day 
has  been  found  to  be  fatal  to  it.  The  facts  established  accord- 
ingly show  it  to  be  a  somewhat  delicate  parasite. 

As  stated  above,  the  organism  occurs  in  the  exudate  in  the 
meninges  and  in  the  cerebro-spinal  fluid,  and  it  can  usually  be 
obtained  by  lumbar  puncture.  In  acute  cases,  especially  in  the 


EPIDEMIC   CEREBRO-SPINAL   MENINGITIS     215 

earlier  stages,  it  is  usually  abundant ;  but  in  the  later  stages  of 
cases  of  more  sub-acute  character,  its  detection  may  be  a  matter 
of  difficulty,  and  only  a  few  examples  may  be  found  after  a 
prolonged  search ;  in  extremely  acute  cases  also  the  organism 
may  be  difficult  to  demonstrate.  In  most  cases  the  disease  is 
practically  restricted  to  the  nervous  system,  but  occasionally 
complications  occur,  and  in  these  the  organism  may  sometimes 
be  found.  It  has  been  observed,  for  example,  in  arthritis,  peri- 
carditis, pneumonic  patches  in  the  lung,  and  in  other  inflam- 
matory conditions  associated  with  the  disease.  In  a  small 
proportion  of  cases  it  has  been  obtained  from  the  blood  during 
life,  but  cultures  in  most  instances  give  negative  results. 

Experimental  inoculation  shows  that  the  ordinary  laboratory 
animals  are  relatively  insusceptible  to  this  organism.  An 
inflammatory  condition  may  be  produced  in  guinea-pigs  by 
intra-peritoneal  injection,  but  large  quantities  of  cultures  must 
be  used,  and  none  of  the  characteristic  lesions  found  in  the 
human  subject  are  reproduced.  The  intra-peritoneal  injection 
of  the  cerebro-spinal  fluid  or  of  cultures  in  mice  is  frequently 
followed  by  death,  the  cocci  being  found  in  the  exudate  and  even 
in  the  blood.  Flexner  has  shown  that  cerebro  spinal  meningitis 
may  be  produced  in  monkeys  by  injections  of  the  organism  into 
the  spinal  canal.  In  such  experiments  the  organism  extends 
upwards  to  the  brain,  and  produces  meningitis  within  a  very 
short  time.  The  resulting  lesions,  both  as  regards  their  dis- 
tribution and  general  characters,  and  also  as  regards  the 
histological  changes,  resemble  the  disease  in  the  human  subject. 
Even  these  animals,  however,  are, .in  comparison  with  man, 
relatively  insusceptible,  as  a  considerable  amount  of  culture  has 
to  be  injected. 

Many  questions  of  great  importance  with  regard  to  the 
spread  of  the  disease  still  require  further  investigation.  The 
organism  has  been  obtained  by  culture  from  the  throat  and 
nasal  cavities  of  those  suffering  from  the  disease  in  a  consider- 
able number  of  instances.  It  has  also  been  obtained  from  the 
same  positions  in  healthy  individuals,  during  an  epidemic  of 
the  disease.  In  some  epidemics  also  a  pharyngitis  has  been 
found  to  occur,  and  the  organism  has  been  obtained  from 
the  affected  fauces.  The  general  opinion  is  that  the  organism 
spreads  by  means  of  the  lymphatics  from  the  pharynx  or 
nose  to  the  base  of  the  brain,  but  a  spread  by  means  of  the 
blood  stream  cannot  be  excluded,  and  infection  by  the  alimentary 
canal  has  also  been  suggested.  Flexner  in  his  experiments 
found  that  when  the  organism  was  injected  into  the  spinal 


216  THE   ACUTE   PNEUMONIAS 

canal  marked  congestion  and  inflammatory  change  in  the 
nasal  mucous  membrane  followed,  and  in  this  position  he  was 
able  to  find  a  Gram-negative  diplococcus ;  he  was,  however, 
unable  to  recover  the  diplococcus  intracellularis  in  culture  from 
this  situation.  These  results  would  seem  to  indicate  that  the 
organism  might  spread  from  the  brain  to  the  nasal  cavity,  but  if 
this  be  so,  it  also  follows  that  an  extension  may  take  place  in 
the  reverse  direction.  On  the  whole  the  evidence  at  present 
tends  to  show  that  the  entrance  of  the  organism  into  the  body 
is  by  the  naso-pharynx,  and  that  this  usually  results  by 
inhalation  of  the  organism  distributed  in  fine  particles  of 
expectoration,  etc.  In  fact,  as  regards  the  mode  and  conditions 
of  infection,  an  analogy  would  appear  to  hold  between  this 
disease  and  influenza. 

Apart  from  the  epidemic  form  of  the  disease,  cases  of  sporadic 
nature  also  occur,  in  which  the  lesions  are  of  the  same  nature, 
and  in  which  the  diplococcus  intracellularis  is  present.  The 
facts  stated  would  indicate  that  the  origin  and  spread  of  the 
disease  in  the  epidemic  form  depend  on  certain  conditions  which 
produce  an  increased  virulence  of  the  organism.  We  are,  however, 
as  yet  entirely  ignorant  as  to  what  these  conditions  may  be.  In 
simple  posterior  basal  meningitis  in  children,  a  diplococcus  is 
present,  as  described  by  Still,  which  has  the  same  microscopic 
and  cultural  characters  as  the  diplococcus  intracellularis ;  it  has 
been  regarded  as  probably  an  attenuated  variety  of  the  latter. 
Recently,  however,  Houston  and  Rankin  have  found  that  the 
serum  of  a  patient  suffering  from  epidemic  meningitis  does  not 
exert  the  same  opsonic  and  agglutinative  effects  on  the  diplococcus 
of  basal  meningitis  as  on  the  diplococcus  intracellularis;  and 
this  result  points  to  the  two  organisms  being  distinct,  though 
closely  allied,  species. 

An  agglutination  reaction  towards  the  diplococcus  intracellu- 
laris is  given  by  the  serum  of  patients  suffering  from  the  disease, 
where  life  is  prolonged  for  a  sufficient  length  of  time,  but  the 
degree  of  the  reaction  does  not  possess  much  clinical  significance. 
It  usually  appears  about  the  fourth  day,  when  the  serum  may 
give  a  positive  reaction  in  a  dilution  of  1  : 50 ;  at  a  later  stage 
it  has  been  observed  in  so  great  a  dilution  as  1  : 1000.  There  is 
thus  no  doubt  that  anti-substances  are  produced  in  epidemic 
meningitis  as  in  other  diseases,  and  this  is  also  found  to  be  the 
case  on  inoculation  of  animals  with  pure  cultures.  Attempts 
had  been  made  to  obtain  an  anti-serum,  and  a  certain  measure  of 
success  has  been  obtained  so  far  as  experimental  results  are 
concerned.  Flexner  obtained  such  a  serum  from  a  goat,  and 


EPIDEMIC   CEREBRO-SPINAL   MENINGITIS     217 

found  that  it  had  a  certain  protective  effect  in  guinea-pigs  and 
monkeys  against  infection  by  the  organism,  but,  on  the  whole, 
better  results  were  obtained  with  the  serum  of  inoculated 
monkeys.  As  yet  no  important  applications  towards  the 
treatment  of  the  disease  have  been  effected. 

In  the  nasal  cavity  there  occur  other  diplococci  which  have  a 
close  resemblance  to  the  diplococous  intracellularis.  These  occur 
in  the  healthy  state  but  are  especially  abundant  in  catarrhal 
conditions  ;  of  these  the  diplococcus  catarrhalis  has  the  closest 
resemblance  to  the  diplococcus  intracellularis.  In  addition  to 
occurring  in  health  this  organism  has  also  been  found  in  large 
numbers  in  epidemic  catarrh.  Its  microscopic  appearances  are 
practically  similar  to  those  described  above,  and  it  also  occurs 
within  leucocytes.  Its  colonies  on  serum  agar  are  more  opaque 
than  those  of  the  diplococcus  intracellularis,  and  they  have  a 
tough  consistence,  so  that  they  are  sometimes  removed  en  masse 
by  the  platinum  needle.  The  organism  grows  on  gelatin  at 
20°  C.  without  liquefying  the  medium,  and  it  has  none  of  the 
fermentative  properties  described  above  as  belonging  to  the 
diplococcus  intracellularis.  Other  species  of  Gram  -  negative 
micrococci  have  also  been  isolated,  and  a  Gram-positive  diplo- 
coccus called  the  diplococcus  crassus  is  of  common  occurrence  • 
this  organism  is  rather  larger  than  the  diplococcus  intracellularis, 
and  especially  in  sub-cultures  may  tend  to  assume  staphylo- 
coccal  forms.  It  is  thus  evident  that  the  nasal  cavity  is  the 
common  habitat  for  a  number  of  closely  allied  diplococci,  and 
that  the  identification  of  any  suspected  organism  as  the  diplo- 
coccus intracellularis  can  only  be  effected  by  cultivation  tests. 

Apart  from  the  epidemic  form  of  the  disease,  meningitis  may 
be  produced  by  almost  any  of  the  organisms  described  in  the 
previous  chapter,  as  associated  with  inflammatory  conditions. 
A  considerable  number  of  cases,  especially  in  children,  are  due 
to  the  pneumococcus.  In  many  instances  where  no  other  lesions 
are  present  the  extension  is  by  the  Eustachian  tube  to  the  middle 
ear.  In  other  cases  the  path  of  infection  is  from  some  other 
lesion  by  means  of  the  blood  stream.  This  organism  also  infects 
the  meninges  not  infrequently  in  lobar  pneumonia,  and  in  some 
cases  with  head  symptoms  we  have  found  it  present  where  there 
was  merely  a  condition  of  congestion.  The  pneumobacillus  also 
has  been  found  in  a  few  cases.  Meningitis  is  not  infrequently 
produced  by  streptococci,  especially  when  middle  ear  disease  is 
present,-  less  frequently  by  one  of  the  staphylococci ;  occasionally 
more  than  one  organism  may  be  concerned.  In  meningitis 
following  influenza  the  influenza  bacillus  has  been  found  in  a 


218  THE   ACUTE   PNEUMONIAS 

few  instances,  but  sometimes  the  pneumococcus  is  the  causal 
agent ;  and  in  tubercular  meningitis  the  tubercle  bacillus  of 
course  is  present,  especially  in  the  nodules  along  the  sheaths  of 
the  vessels.  An  invasion  of  the  meninges  by  the  anthrax 
bacillus  occurs,  but  is  a  rare  condition ;  it  is  attended  by  diffuse 
haemorrhage  in  the  subarachnoid  space. 

In  conclusion  here  it  may*be  stated  that  mixed  infections 
may  occur  in  meningitis.  Thus  the  pneumococcus  has  been 
found  associated  with  the  tubercle  bacillus  and  also  with  the 
diplococcus  intracellularis.  Further,  in  infection  with  the  latter, 
Gram-negative  bacilli  of  a  diphtheroid  appearance  have  also  been 
observed ;  the  significance  of  these  is  unknown. 


CHAPTER   VIII. 
GONORRHOEA,   SOFT   SORE,    SYPHILIS. 

GONORRHOEA. 

Introductory. — The  micrococcus  now  known  to  be  the  cause  of 
gonorrhoea,  and  now  called  the  gonococcus,  was  first  described 
by  Neisser,  who  in  1879  gave  an  account  of  its  microscopical 
characters  as  seen  in  the  pus  of  gonorrhoeal  affections,  both  of 
the  urethra  and  of  the  conjunctiva.  He  considered  that  this 
organism  was  peculiar  to  the  disease,  and  that  its  characters 
were  distinctive.  Later  it  was  successfully  isolated  and  cultivated 
on  solidified  blood  serum  by  Bumm  and  others.  Its  characters 
have  since  been  minutely  studied,  and  by  inoculations  of  cultures 
on  the  human  subject  its  causal  relationship  to  the  disease  has 
been  conclusively  established. 

The  Gonococcus. — Microscopical  Characters. — The  organism 
of  gonorrhoea  is  a  small  micrococcus  which  usually  is  seen  in  the 
diplococcus  form,  the  adjacent  margins  of  the  two  cocci  being 
flattened,  or  even  slightly  concave,  so  that  between  them  there  is 
a  small  oval  interval  which  does  not  stain.  An  appearance  is 
thus  presented  which  has  been  compared  to  that  of  two  beans 
placed  side  by  side  (vide  Fig.  74).  When  division  takes  place  in 
the  two  members  of  a  diplococcus,  a  tetrad  is  formed,  which, 
however,  soon  separates  into  two  sets  of  diplococci — that  is  to 
say,  arrangement  as  diplococci  is  much  commoner  than  as  tetrads. 
Cocci  in  process  of  degeneration  are  seen  as  spherical  elements 
of  varying  size,  some  being  considerably  swollen. 

These  organisms  are  found  in  large  numbers  in  the  pus  of 
acute  gonorrhoea,  both  in  the  male  and  female,  and  for  the  most 
part  are  contained  within  the  leucocytes.  In  the  earliest  stage, 
when  the  secretion  is  glairy,  a  considerable  number  are  lying 
free,  or  are  adhering  to  the  surface  of  desquamated  epithelial 

219 


220         GONORRHCEA,    SOFT   SORE,    SYPHILIS 


cells,  but  when  it  becomes  purulent  the  large  proportion  within 
leucocytes  is  a  very  striking  feature.  In  the  leucocytes  they  lie 
within  the  protoplasm,  especially  superficially,  and  are  often  so 

numerous  that  the  leuco- 
cytes appear  to  be  filled 
with  them,  and  their  nuclei 
are  obscured.  As  the  dis- 
ease becomes  more  chronic, 
the  gonococci  gradually 
become  diminished  in 
number,  though  even  in 
long-standing  cases  they 
may  still  be  found  in  con- 
siderable numbers.  They 
are  also  present  in  the 
purulent  secretion  of  gon- 
orrhceal  conjunctivitis,  also 
in  various  parts  of  the 

female  genital  organs  when 
FIG.  74.— Portion  of  film  of  gonorrhceal  pus,  thege  partg  are  ^  geat  of 

showing  the  characteristic   arrangement    ,  •,        -,   •    £     ,. 

of  the  gonococci  within  leucocytes.  tmf  gonorrhoeal  infection, 

Stained  with  fuchsin.  x  1000.  and  they  have  been  found 

in  some  cases  in  the  second- 
ary infections  of  the  joints  in  the  disease,  as  will  be  described 
below. 

Staining. — The  gonococcus  stains  readily  and  deeply  with  a 
watery  solution  of  any  of  the  basic  aniline  dyes — methylene-blue, 
fuchsin,  etc.  It  is,  however,  easily  decolorised,  and  it  completely 
loses  the  stain  by  Gram's  method — an  important  point  in  the 
microscopical  examination. 

Cultivation  of  the  Gonococcus. — This  is  attended  with  some 
difficulty,  as  the  suitable  media  and  conditions  of  growth  are 
somewhat  restricted.  The  most  suitable  media  are  solidified 
blood  serum  (especially  human  serum  and  rabbit's  serum), 
"blood  agar,"  and  Wertheim's  medium,  which  consists  of  one 
part  of  fluid  serum  added  to  two  parts  of  liquefied  agar  at  a 
temperature  of  40°  C.  and  then  allowed  to  solidify  by  cooling. 
The  serum  may  be  obtained  from  the  blood  of  the  human 
placenta ;  pleuritic  or  other  effusion  may  also  be  used. 
Growth  takes  place  best  at  the  temperature  of  the  body,  and 
ceases  altogether  at  25°  C.  Cultures  are  obtained  by  taking 
some  pus  on  the  loop  of  the  platinum  needle  and  inoculating 
one  of  the  media  mentioned  by  leaving  minute  quantities  here 
and  there  on  the  surface.  The  medium  may  be  used  either  as 


CULTIVATION   OF   GONOCOCCUS 


221 


ordinary  "sloped  tubes"  or  as  a  thin  layer  in  a  Petri's  capsule. 
The  young  colonies  are  visible  within  forty-eight  hours,  and  often 
within  twenty-four  hours.  They  appear  around  the  points  of 
inoculation  as  small  semi-transparent  discs  of  irregularly  rounded 
shape,  the  margin  being  undulated  and  sometimes  showing  small 
processes.  The  colonies  vary  somewhat  in  size  and  tend  to 
remain  more  or  less  separate.  They  generally  reach  their 
maximum  size  on  the  fourth  or  fifth  day,  and  are  usually  found 
to  be  dead  on  the  ninth  day,  sometimes  earlier.  On  the 
medium  of  Wertheim  the  period  of  active  growth  and  the 
duration  of  life  are  some- 
what longer.  Even  if 
impurities  are  present,  pure 
sub-cultures  can  generally 
be  obtained  by  the  above 
method  from  colonies  of 
the  gonococcus  which  may 
be  lying  separate.  In  the 
early  stage  of  the  disease 
the  organism  is  present  in 
the  male  urethra  in  prac- 
tically pure  condition,  and 
if  the  meatus  of  the  urethra 
be  sterilised  by  washing 
with  weak  solution  of  cor- 
rosive sublimate  and  then 
with  absolute  alcohol,  and  FIG.  75.—  Gonococci,  from  a  pure  culture 
the  material  for  inoculation  on  blood  agar  of  twenty -four  hours' 
V»P  Pvnrp^pH  from  thp  growth.  Some  already  are  beginning  to 
be  expressed  trom  tne  Jhow  the  swollen  appearance  common  in 
deeper  part  of  the  urethra,  oljer  cuitures. 

cultures  may  often  be  ob-     Stained  with  carbol-thionin-blue.      x  1000. 
tained    which     are     pure 

from  the  first.  By  successive  sub-cultures  at  short  intervals, 
growth  may  be  maintained  indefinitely,  and  the  organism 
gradually  flourishes  more  luxuriantly.  In  culture  the  organisms 
have  similar  microscopic  characters  to  those  described  (Fig.  75), 
but  show  a  remarkable  tendency  to  undergo  degeneration, 
becoming  swollen  and  of  various  sizes,  and  staining  very 
irregularly.  Degenerated  forms  are  seen  even  on  the  second 
day,  whilst  in  a  culture  four  or  five  days  old  comparatively  few 
normal  cocci  may  be  found.  The  less  suitable  the  medium  the 
more  rapidly  does  degeneration  take  place. 

On  ordinary  agar  and  on  glycerin  agar  growth  does  not  take 
place,  or  is  so  slight  that  these  media  are  quite  unsuitable  for 


222         GONORRHOEA,    SOFT   SORE,    SYPHILIS 

purposes  of  culture.     The  organism  does  not  grow  on  gelatin,1 
potato,  etc. 

Plate-Cultures. — The  following  ingenious  method  of  plate-culture  was 
introduced  by  Wertheim  for  the  culture  of  the  gonococcus.  The  medium 
of  culture  is  a  mixture  of  human  blood  serum  and  of  ordinary  agar  (2 
per  cent)  in  equal  parts.  The  serum,  in  a  fluid  and  sterile  condition,  is 
put  in  suitable  quantities  into  two  or  three  test  tubes  and  brought  to  a 
temperature  of  40°  C.  These  are  then  successively  inoculated  with  the 
pus  or  other  material  in  the  same  manner  as  gelatin  tubes  for  ordinary 
plates  (vide  p.  52).  To  each  tube  is  added  an  equal  part  of  ordinary 
agar  which  has  been  thoroughly  liquefied  by  heating  and  allowed  to 
cool  also  to  40°  C.  The  mixture  is  then  thoroughly  shaken  up  and 
quickly  poured  out  on  a  plate  or  Petri's  dish  and  allowed  to  solidify, 
the  plates  being  then  incubated  at  a  temperature  of  37°  C.  The  colonies 
of  the  gonococcus  are  just  visible  in  twenty-four  hours,  and  are  seen 
both  in  the  substance  of  the  medium  and  on  the  surface.  The  deep 
colonies  when  examined  with  a  lens  are  minute  and  slightly  nodulated 
spheres,  sometimes  showing  little  processes,  whilst  those  on  the  surface 
are  thin  discs  of  larger  diameter  with  wavy  margin  and  rather  darker 
centre.  In  this  way  the  gonococcus  may  be  separated  from  fluids  which 
are  contaminated  with  a  considerable  number  of  other  organisms. 

Relations  to  the  Disease.  —  The  gonococcus  is  invariably 
present  in  the  urethral  discharge  in  gonorrhoea,  and  also  in 
other  parts  of  the  genital  tract  when  these  are  the  seat  of  true 
gonorrhoeal  infection.  Its  presence  in  these  different  positions 
has  been  demonstrated  not  only  by  microscopic  examination 
but  also  by  culture.  From  the  description  of  the  conditions  of 
growth  in  culture,  it  will  be  seen  that  a  life  outside  the  body 
in  natural  conditions  is  practically  impossible — a  statement 
which  corresponds  with  the  clinical  fact  that  the  disease  is 
always  transmitted  directly  by  contagion.  Inoculations  of  pure 
cultures  on  the  urethra  of  lower  animals,  and  even  of  apes,  is 
followed  by  no  effect,  but  a  similar  statement  can  be  made  with 
regard  to  inoculations  of  gonorrhoeal  pus  itself.  In  fact, 
hitherto  it  has  been  found  impossible  to  reproduce  the  disease  by 
any  means  in  the  lower  animals.  On  a  considerable  number  of 
occasions  inoculations  of  pure  cultures  have  been  made  on  the 
human  urethra,  both  in  the  male  and  female,  and  the  disease, 
with  all  its  characteristic  symptoms,  has  resulted.  (Such 
experiments  have  been  performed  independently  by  Bumm, 
Steinschneider,  Wertheim,  and  others.)  The  causal  relationship 
of  the  organism  to  the  disease  has  therefore  been  completely 

1  Turro  has  announced  that  he  lias  cultivated  the  gonococcus  on  acid 
gelatin,  i.e.  ordinary  peptone  gelatin  which  has  not  been  neutralised.  We 
have  failed  to  obtain  any  growth  of  the  gonococcus  on  this  medium,  even 
when  inoculation  was  made  from  a  vigorous  growth  on  blood  agar. 


DISTRIBUTION   OF   GONOCOCCUS  223 

established,  and  it  is  interesting  to  note  how  the  conditions  of 
growth  and  the  pathogenic  effects  of  the  organism  agree  with 
the  characters  of  the  natural  disease. 

Intraperitoneal  injections  of  pure  cultures  of  the  gonococcus  in  white 
mice  produce  a  localised  peritonitis  with  a  small  amount  of  suppuration, 
the  organisms  being  found  in  large  numbers  in  the  leucocytes  (Wertheim). 
They  also  penetrate  the  peritoneal  lining  and  are  found  in  the  sub- 
endothelial  connective  tissue,  but  they  appear  to  have  little  power  of 
proliferation,  they  soon  disappear,  and  the  inflammatory  condition  does 
not  spread.  Injection  of  pure  cultures  into  the  joints  of  rabbits,  dogs, 
and  guinea-pigs  causes  an  acute  inflammation,  which,  however,  soon 
subsides,  whilst  the  gonococci  rapidly  die  out  ;  a  practically  similar 
result  is  obtained  when  dead  cultures  are  used.  These  experiments  show 
that  while  the  organism,  when  present  in  large  numbers,  can  produce  a 
certain  amount  of  inflammatory  change  in  these  animals,  it  has  little  or 
no  power  of  multiplying  and  spreading  in  their  tissues. 

Toxin  of  the  Gonococcus. — De  Christmas  cultivated  the  gonococcus  in 
a  mixture  of  one  part  of  ascitic  fluid  and  three  parts  of  bouillon,  and 
has  found  that  the  fluid  after  twelve  days'  growth  has  toxic  properties. 
At  this  period  all  the  organisms  are  dead  ;  such  a  fluid  constitutes  the 
"  toxin."  The  toxic  substances  are  precipitated  along  with  the  proteids 
by  alcohol,  and  the  precipitate  after  being  desiccated  possesses  the  toxic 
action.  In  young  rabbits  infection  of  the  toxin  produces  suppuration  ; 
this  is  well  seen  in  the  anterior  chamber  of  the  eye,  where  hypopyon 
results.  The  most  interesting  point,  however,  is  with  regard  to  its 
action  on  mucous  surfaces  ;  for,  while  in  the  case  of  animals  it  produces 
no  effect,  its  introduction  into  the  human  urethra  causes  acute  catarrh, 
attended  with  purulent  discharge.  He  found  that  no  tolerance  to  the 
toxin  resulted  after  five  successive  injections  at  intervals.  In  a  more 
recent  publication  he  points  out  that  the  toxin  on  intracerebral  injec- 
tion has  marked  effects  ;  he  also  claims  to  have  produced  an  antitoxin. 
He  states  that  the  toxin  diffuses  out  in  the  culture  medium,  and  does 
not  merely  result  from  disintegration  of  the  organisms.  This  has,  how- 
ever, been  called  in  question  by  other  investigators. 

Distribution  in  the  Tissues. — The  gonococcus  having  been 
thus  shown  to  be  the  direct  cause  of  the  disease,  some  additional 
facts  may  be  given  regarding  its  presence  both  in  the  primary 
and  secondary  lesions.  In  the  human  urethra  the  gonococci 
penetrate  the  mucous  membrane,  passing  chiefly  between  the 
epithelial  cells,  causing  a  loosening  and  desquamation  of  many 
of  the  latter  and  inflammatory  reaction  in  the  tissues  below, 
attended  with  great  increase  of  secretion.  There  occurs  also 
a  gradually  increasing  emigration  of  leucocytes,  which  take  up  a 
large  number  of  the  organisms.  The  organisms  also  penetrate 
the  subjacent  connective  tissue,  and  are  especially  found  along 
with  extensive  leucocytic  emigration  around  the  Iacuna3.  Here 
also  many  are  contained  within  leucocytes.  Even,  however, 
when  the  gonococci  have  disappeared  from  the  urethral  dis- 
charge, they  may  still  be  present  in  the  deeper  part  of  the 


224         GONORRHCEA,    SOFT    SORE,    SYPHILIS 

mucous  membrane  of  the  urethra,  possibly  also  in  the  prostate, 
and  may  thus  be  capable  of  producing  infection.  The  prostatic 
secretion  may  sometimes  be  examined  by  making  pressure  on 
the  prostate  from  the  rectum  when  the  patient  has  almost  emptied 
his  bladder,  the  secretion  being  afterwards  discharged  along  with 
the  remaining  urine.  (Foulerton.)  In  acute  gonorrhoea  there 
is  often  a  considerable  degree  of  inflammatory  affection  of  the 
prostate  and  vesiculse  seminales,  but  whether  these  conditions  are 
always  due  to  the  presence  of  gonococci  in  the  affected  parts 
we  have  not  at  present  the  data  for  determining.  A  similar 
statement  also  applies  to  the  occurrence  of  orchitis  and  also  of 
cystitis  in  the  early  stage  of  gonorrhoea.  Gonococci  have, 
however,  been  obtained  in  pure  culture  from  peri-urethral  abscess 
and  from  epididymitis  :  it  is  likely  that  the  latter  condition, 
when  occurring  in  gonorrhoea,  is  usually  due  to  the  actual 
presence  of  gonococci.  During  the  more  chronic  stages  other 
organisms  may  appear  in  the  urethra,  aid  in  maintaining  the 
irritation,  and  may  produce  some  of  the  secondary  results. 
The  bacillus  coli,  the  pyogenic  cocci,  etc.,  are  often  present,  and 
may  extend  along  the  urethra  to  the  bladder  and  set  up  cystitis, 
though  in  this  they  may  be  aided  by  the  passage  of  a  catheter. 
It  may  be  mentioned  here  that  Wertheim  cultivated  the 
gonococcus  from  a  case  of  chronic  gonorrhoea  of  two  years' 
standing,  and  by  inoculation  on  the  human  subject  proved  it  to 
be  still  virulent. 

In  the  disease  in  the  female,  gonococci  are  almost  invariably 
present  in  the  urethra,  the  situation  affected  next  in  frequency 
being  the  cervix  uteri.  They  do  not  appear  to  infect  the  lining 
epithelium  of  the  vagina  of  the  adult  unless  some  other  abnormal 
condition  be  present,  but  they  do  so  in  the  gonorrhoeal  vulvo- 
vaginitis  of  young  subjects.  They  have  also  been  found  in 
suppurations  in  connection  with  Bartholini's  glands,  and  some- 
times produce  an  inflammatory  condition  of  the  mucous  membrane 
of  the  body  of  the  uterus.  They  may  also  pass  along  the 
Fallopian  tubes  and  produce  inflammation  of  the  mucous  mem- 
brane there.  From  the  pus  in  cases  of  pyosalpinx  they  have 
been  cultivated  -in  a  considerable  number  of  cases.  According 
to  the  results  of  various  observers  they  are  present  in  one  out 
of  four  or  five  cases  of  this  condition,  usually  unassociated  with 
other  organisms.  Further,  in  a  large  proportion  of  the  cases  in 
which  the  gonococcus  has  not  been  found  no  organisms  of  any 
kind  have  been  obtained  from  the  pus,  and  in  these  cases  the 
gonococci  may  have  been  once  present  and  have  subsequently 
died  out.  Lastly,  they  may  pass  to  the  peritoneum  and  produce 


DISTRIBUTION   OF   GONOCOCCUS  225 

peritonitis,  which  is  usually  of  a  local  character.  It  is  chiefly  to 
the  methods  of  culture  supplied  by  Wertheim  that  we  owe  our 
extended  knowledge  of  such  conditions. 

In  gonorrhoeal  conjunctivitis  the  mode  in  which  the  gonococci 
spread  through  the  epithelium  to  the  subjacent  connective 
tissue  is  closely  analogous  to  what  obtains  in  the  case  of  the 
urethra.  Their  relation  to  the  leucocytes  in  the  purulent 
secretion  is  also  the  same.  Microscopic  examination  of  the 
secretion  alone  in  acute  cases  often  gives  positive  evidence,  and 
pure  cultures  may  be  readily  obtained  on  blood-agar.  As  the 
condition  becomes  more  chronic  the  gonococci  are  less  numerous 
and  a  greater  proportion  of  other  organisms  may  be  present. 

Relations  to  Joint  A/ections,  etc. — The  relations  of  the  gono- 
coccus  to  the  sequelae  of  gonorrhoea  form  a  subject  of  great 
interest  and  importance,  and  the  application  of  recent  methods  of 
examination  shows  that  the  organism  is  much  more  frequently 
present  in  such  conditions  than  the  earlier  results  indicated. 
The  following  statements  may  be  made  with  regard  to  them. 
First,  in  a  considerable  number  of  cases  of  arthritis  following 
gonorrhoea  the  gonococcus  has  been  found  microscopically,  and 
pure  cultures  have  been  obtained,  e.g.  by  Neisser,  Lang, 
Bordoni-Uffreduzzi,  and  many  others.  A  similar  statement 
applies  to  inflammation  of  the  sheaths  of  tendons  following 
gonorrhoea.  Secondly,  in  a  large  proportion  of  cases  no  organ- 
isms have  been  found.  It  is,  however,  possible  that  in  a  number 
of  these  the  gonococci  may  have  been  present  in  the  synovial 
membrane,  as  it  has  been  observed  that  they  may  be  much  more 
numerous  in  that  situation  than  in  the  fluid.  Thirdly,  in  some 
cases,  especially  in  those  associated  with  extensive  suppuration, 
occasionally  of  a  pysemic  nature,  various  pyogenic  cocci  have 
been  found  to  be  present.  In  the  instances  in  which  the  gono- 
coccus has  been  found  in  the  joints,  the  fluid  present  has  usually 
been  described  as  being  of  a  whitish-yellow  tint,  somewhat 
turbid,  and  containing  shreds  of  fibrin-like  material,  sometimes 
purulent  in  appearance.  In  one  case  Bordoni-Uffreduzzi  culti- 
vated the  gonococcus  from  a  joint-affection,  and  afterwards 
produced  gonorrhoea  in  the  human  subject  by  inoculating  with 
the  cultures  obtained.  In  another  case  in  which  pleurisy  was 
present  along  with  arthritis  the  gonococcus  was  cultivated  from 
the  fluid  in  the  pleura!  cavity.  The  existence  of  a  gonorrhoeal 
endocarditis  has  been  established  by  recent  observations.  Cases 
apparently  of  this  nature  occurring  in  the  course  of  gonorrhoea 
had  been  previously  described,  but  the  complete  bacteriological 
test  has  now  been  satisfied  in  several  instances.  In  one  case 
15 


226         GONORRHOEA,    SOFT   SORE,    SYPHILIS 

Lenhartz  produced  gonorrhoea  in  the  human  subject  by 
inoculation  with  the  organisms  obtained  from  the  vegetations. 
That  a  true  gonorrhoeal  septicaemia  may  also  occur  has  also  been 
established,  cultures  of  the  gonococcus  having  been  obtained 
from  the  blood  during  life  on  more  than  one  occasion  (Thayer 
and  Blumer,  Thayer  and  Lazear,  Ahmann). 

Methods  of  Diagnosis. — For  microscopical  examination  dried 
films  of  the  suspected  pus,  etc.,  may  be  stained  by  any  of  the 
simple  solutions  of  the  basic  aniline  stains.     We  prefer  methy- 
lene-  or  thionin-blue,  as  they  do  not  overstain,  and  the  films  do 
not  need  to  be  decolorised.     Staining  for  one  minute  is  sufficient. 
It  is  also  advisable  to  stain  by  Gram's  method,  and  it  is  a  good 
plan  to  put  at  one  margin  of  the  cover-glass  a  small  quantity  of 
culture  of  staphylococcus  aureus  if  available,  in  order  to  have  a 
standard  by  which  to  be  certain  that  the  supposed  gonococci  are 
really  decolorised.     Regarding  the  value  of  microscopic  examina- 
tion alone,  we  may  say  that  the  presence  of  a  large  number  of 
micrococci  in  a  urethral  discharge  having  the  characters,  position, 
and  staining  reactions  described  above,  is  practically  conclusive 
that  the  case  is  one  of  gonorrhoea.     There  is  no  other  condition 
in  which  the  sum  total  of  the  microscopical  characters  is  present. 
We  consider  that  it  is  sufficient  for  purposes  of  clinical  diagnosis, 
and  therefore  of  great  value ;  in  the  acute  stage  a  diagnosis  can 
thus  be  made  earlier  than  by  any  other  method.     The  mistake 
of  confusing  gonorrhoea  with  such  conditions  as  a  urethral  chancre 
with  urethritis,  will  also  be  avoided.     Even  in  chronic  cases  the 
typical  picture  is  often  well  maintained,  and  microscopic  examina- 
tion alone  may  give  a  definite  positive  result.    When  other  organ- 
isms are  present,  and  especially  when  the  gonococci  are  few  in 
number,  it  is  difficult,  and  in  some  cases  impossible,  to  give  a 
definite  opinion,  as  a  few  gonococci  mixed  with  other  organisms 
cannot  be  recognised  with  certainty.     This  is  often  the  condition 
in  chronic  gonorrhoea  in  the  female.     Microscopic  examination, 
therefore,  though  often  giving  positive  results,  will  sometimes  be 
inconclusive.     As  regards  lesions   in  other  parts  of   the  body 
microscopic  examination  alone  is  quite  insufficient ;  it  is  practi- 
cally impossible,  for  example,  to  distinguish  by  this  means  the 
gonococcus   from  the  diplococcus  intracellularis  of  meningitis. 
Cultures  alone  supply  the  absolute  test,  and  when  the  organism 
is   present    in   an   apparent    condition    of   purity,    Wertheim's 
medium  or   blood -agar  should   be  used.       If  other  organisms 
are  present,  we  are   practically  restricted  to  Wertheim's  plate 
method. 


SOFT   SORE 


227 


SOFT  SOKE. 


The   bacillus    of  soft   sore   was 
in  1889,   who   found   it  in  the 
ulcerated    surface ;    and   later, 
appearance   and    distribution  as  seen 


first   described  by   Ducrey 
purulent   discharge   from  the 
a    1892,   Unna   described    its 
sections  through  the 


sores.  The  statements  of  these  observers  regarding  the  presence 
and  characters  of  this  organism  have  been  fully  confirmed  by 
other  observers. 

Microscopical  Characters. — The  organism  occurs  in  the  form 
of  minute  oval  rods  measuring  about  1  "5  /x  in  length,  and  *5  //, 
in  thickness  (Fig.  76).  It  is  found  mixed  with  other  organisms 
in  the  purulent  discharge 
from  the  surface,  and  is 
chiefly  arranged  in  small 
groups  or  in  short  chains. 
When  studied  in  sections 
through  the  ulcer  it  is 
found  in  the  superficial 
part  of  the  floor,  but  more 
deeply  situated  than  other 
organisms,  and  may  be 
present  in  a  state  of  pur- 
ity amongst  the  leucocytic 
infiltration.  In  this  posi- 
tion it  is  usually  arranged 
in  chains  which  may  be 
of  considerable  length, 
and  which  are  often  seen 
lying  in  parallel  rows  be- 
tween the  cells.  The 
bacilli  chiefly,  occur  in 
the  free  condition,  but  occasionally  a  few  may  be  contained 
within  leucocytes. 

There  is  no  doubt  that  in  many  cases  the  organism  is 
present  in  the  buboes  in  a  state  of  purity ;  it  has  been  found 
there  by  microscopic  examination,  and  cultures  have  also  been 
obtained  from  this  source.  The  negative  results  of  some 
observers  are  probably  due  to  the  organism  having  died  off. 
On  the  whole  the  evidence  goes  to  show  that  the  ordinary 
bubo  associated  with  soft  sore  is  to  be  regarded  as  another  lesion 
produced  by  Ducrey's  bacillus.  Sometimes  the  ordinary  pyogenic 
organisms  become  superadded. 

This  bacillus  takes  up  the  basic  aniline  stains  fairly  readily, 


FIG.  76. — Film  preparation  of  pus  from  soft 
chancre,  showing  Ducrey's  bacillus,  chiefly 
arranged  in  pairs  ;  stained  with  carbol- 
fuchsin  and  slightly  decolorised,  x  1500. 


228         GONORRHOEA,    SOFT   SORE,   SYPHILIS 


but  loses  the  colour  very  rapidly  when  a  decolorising  agent  is 
applied.  Accordingly,  in  film  preparations  when  dehydration 
is  not  required,  it  can  be  readily  stained  by  most  of  the 
ordinary  combinations,  though  Loffler's  or  Kiihne's  methylene- 
blue  solutions  are  preferable,  as  they  do  not  overstain.  In 
sections,  however,  great  care  must  be  taken  in  the  process  of 
dehydration,  and  the  aniline-oil  method  (vide  p.  93)  should  be 
used  for  this  purpose,  as  alcohol  decolorises  the  organism  very 
readily.  A  little  of  the  methylene-blue  or  other  stain  may  be 
with  advantage  added  to  the  aniline- oil  used  for  dehydrating. 

Cultivation. — Although  for  a  long  period  of  time  attempts 
to  obtain  cultures  were  unsuccessful,  success  has  been  attained 

within  recent  years. 

^  +  ^  Benzangon,  Griffon,  and 

Le  Sourd  obtained  pure 
cultures  in  four  cases, 
the  medium  used  being 
a  mixture  of  rabbit's 
blood  and  agar,  in  the 
proportion  of  one  part 

of  the  former  to  two  of 
the  latter.  The  blood 

is  added  to  the  agar  in 
the  melted  condition  at 
45°  C.,  and  the  tubes  are 
then  sloped.  Davis  con- 
firms these  results,  and 
finds  that  another  good 
medium  is  freshly-drawn 
human  blood  distributed 
in  small  tubes ;  this 

method  is  specially  suitable,  as  the  blood  inhibits  the  growth 
of  various  extraneous  organisms.  On  the  solid  medium  (blood- 
agar)  the  growth  appears  in  the  form  of  small  round  globules, 
which  attain  their  complete  development  in  forty-eight  hours, 
having  then  a  diameter  of  1  to  2  mm.;  the  colonies  do  not 
become  confluent.  Microscopic  examination  of  these  colonies, 
which  are  dissociated  with  some  difficulty,  shows  similar  appear- 
ances to  those  observed  when  the  organism  is  in  the  tissues  (Fig. 
77),  but  occasionally  long  undivided  filaments  are  observed  which 
Davis  regards  as  degenerative  forms.  Within  a  comparatively 
short  period  cultures  undergo  marked  degenerative  changes,  and 
great  irregularities  of  form  and  shape  are  to  be  found.  It 

We  are  indebted  to  Dr.  Davis  for  the  use  of  Figs.  76  and  77. 


FIG.  77. — Ducrey's  bacillus  from  a  24-hour 
culture  in  blood-bouillon.      x  1500.1 


SYPHILIS  229 

would  appear  that  a  comparatively  large  amount  of  blood  is 
necessary  for  the  growth  of  this  organism,  and  even  sub-cultures 
on  the  ordinary  media,  including  blood -serum  media,  give 
negative  results.  Inoculation  of  the  ordinary  laboratory  animals 
is  not  attended  by  any  result,  but  it  has  been  found  that  some 
monkeys  are  susceptible,  small  ulcerations  being  produced  by 
superficial  inoculation,  and  in  these  the  organism  can  be  demon- 
strated. Tomasczewski  cultivated  the  organism  for  several 
generations,  and  reproduced  the  disease  by  inoculation  of  the 
human  subject.  The  causal  relationship  of  this  bacillus  must 
therefore  be  considered  as  completely  established,  and  the  con- 
ditions under  which  it  grows  show  it  ta  be  a  strict  parasite — a 
fact  which  is  in  conformity  with  the  known  facts  as  to  the 
transmission  of  the  disease. 

SYPHILIS. 

Up  till  quite  recent  times  practically  nothing  of  a  definite 
nature  was  known  regarding  the  etiology  of  syphilis.  Most 
interest  for  a  long  time  centred  around  the  observations  of 
Lustgarten,  who  in  1884  described  a  characteristic  bacillus, 
both  in  the  primary  sore  and  in  the  lesions  in  internal  organs. 
This  organism  occurred  in  the  form  of  slender  rods,  straight,  or 
slightly  bent,  3  to  4  /A  in  length,  often  forming  little  clusters 
either  within  cells  or  lying  free  in  the  lymphatic  spaces ;  it  took 
up  basic  aniline  dyes  with  some  difficulty,  but  was  much  more 
easily  decolorised  by  acids  than  the  tubercle  bacillus. 

Lustgarten  stained  the  tissues  for  twenty-four  to  forty-eight  hours 
in  aniline-water  solution  of  gentian  violet  ;  and  then,  after  washing 
them  in  alcohol,  placed  them  for  ten  seconds  in  a  1*5  per  cent  solution 
of  permanganate  of  potassium.  They  were  then  treated  with  sulphurous 
acid,  which  removes  the  brown  precipitate  formed,  and  decolorises  the 
sections.  They  were  then  washed  in  water,  dehydrated,  and  mounted. 

Much  controversy  arose  regarding  the  significance  of  this 
bacillus.  Some  considered  it  to  be  the  tubercle  bacillus,  whilst 
others  supposed  that  it  was  the  smegma  bacillus  which  had 
invaded  the  tissues.  The  etiological  relationship  of  the  organism 
to  the  disease  was,  however,  not  generally  accepted,  and  in  view 
of  the  recent  work  on  syphilis,  the  organism  cannot  be  regarded 
as  having  any  pathological  importance. 

Spirochsete  pallida. — An  entirely  new  light  has  been  thrown 
on  the  etiology  of  the  disease  by  the  work  of  Schaudinn  and 
Hoffmann  which  appeared  in  1905.  Since  their  first  publication 
a  great  amount  of  work  has  been  undertaken  in  order  to  test 


230        GONORRHCEA,   SOFT   SORE,    SYPHILIS 

their  conclusions,  and  the  general  result  may  be  said  to  be  of  a 
confirmatory  nature.  These  observers  found  in  certain  cases  an 
organism  to  which  they  gave  the  name  spirochcete  pallida ;  it 
now  also  goes  by  the  name  spironema  pallidum.  As  described 
by  them,  it  is  a  minute  spiral-shaped  organism,  showing  usually 
from  six  to  eight  curves,  though  longer  forms  are  met  with ;  the 
curves  are  small,  comparatively  sharp,  and  regular  (Figs.  78,  79). 
It  may  be  said  to  measure  4-14  p  in  length,  while  it  is  extremely 
thin,  its  thickness  being  only  '25  p.  In  a  fresh  specimen,  say  a 
scraping  from  a  chancre  suspended  in  a  little  salt  solution,  the 
organism  shows  active  movements,  which  are  of  three  kinds — 
rotation  about  the  long  axis,  gliding  movements  to  and  fro, 


FIGS.  78  and  79. — Film  preparations  from  juice  of  hard  chancre  showing 
spirochsete  pallida,— Giemsa's  stain,  x  1000.  (From  preparations  by 
Dr.  A.  MacLennan.) 

and  movements  of  flexion  of  the  whole  body.  The  ends  are 
pointed  and  tapering.  Its  detection  is  comparatively  difficult, 
as  the  organism  is  feebly  refractile,  and  more  difficult  to  see  than 
most  other  organisms ;  the  movement  of  small  particles  in  the 
vicinity,  however,  is  of  assistance  in  finding  it. 

In  ulcerated  syphilitic  lesions  other  organisms  are,  of  course, 
present,  and  not  infrequently  another  spiral  organism,  to  which 
the  name  spirochcete  refringens  has  been  given.  This  organism 
is  usually  somewhat  longer,  and  is  distinctly  thicker  than  the 
spirochsete  pallida.  As  the  name  implies,  it  is  more  highly 
refractile,  and  is  much  more  easily  detected  than  the  latter 
organism ;  its  curves  also  are  opener  and  much  less  regular, 
and  they  vary  in  their  appearance  during  the  movements.  In 
stained  films  (see  p.  107)  the  differences  between  the  organisms 
come  out  more  distinctly,  as  can  be  gathered  from  the  accom- 
panying photograph  (Fig.  81).  The  spirochaete  pallida  by  the 


SYPHILIS  231 

Giemsa  stain  is  coloured  somewhat  faintly,  and  of  reddish  tint, 
whilst  the  regular  spiral  twistings  are  preserved  ;  the  spirochsete 
refringens  shows  flatter,  wave-like  bends,  and,  like  other  organ- 
"isms,  is  stained  of  a  bluish  tint. 
By  using  Loffler's  stain  for  the 
flagella  of  bacteria,  Schaudinn  was 
able  to  demonstrate  a  single  deli- 
cate flagellum  at  each  pole  of  the 
spirochsete  pallida,  while  no  undu-  ^p* 


lating  membrane  could  be  detected ; 
on  the  other  hand,  several  other 
species,  including  the  spirochsete 
refringens,  showed  a  distinct  un- 
dulating membrane.  Two  flagella 
at  one  pole  of  the  spirochsete 
pallida  were  also  seen,  an  appear-  FIG.  80. — Section  of  spleen  from 
ance  which  Schaudinn  thought  a  case  of  congenital  syphilis, 

might  represent  the  commencement        showinS  ,seve^  examPlef  °f 
,.  V        ., r,.      ,  n     .  spirochsete  pallida;  Levaditi  s 

of  longitudinal  fission.  £&aA.     x  1000. 

The  number  of  publications  with 

regard  to  the  distribution  of  the  spirochsete  pallida  is  already 
very  large,  and  a  summary  of  the  results  may  be  given.  In  the 
primary  sore  and  in  the  related  lymphatic  glands,  the  juice  of 
which  can  be  conveniently  obtained  by  means  of  a  hypodermic 

syringe,  the  organism  has  been 
found  in  a  very  large  majority  of 
cases.  It  has  been  also  obtained 
in  the  papular  and  roseolar  erup- 
tions, in  condylomata  and  mucous 
patches  —  in  fact,  one  may  say 
generally,  in  all  the  primary  and 
secondary  lesions.  It  has  been  ob- 
tained from  the  spleen  during  life, 
and  on  a  few  occasions,  e.g.  by 
Schaudinn,  also  from  the  blood 
during  life  in  secondary  syphilis. 

In  the  congenital  form  of  the  disease 

FIG.   81. — Spirochaete    refringens    ,,  .  ,  .     . 

in  film  preparation  from  a  case    the    organism    may   be    present    in 
of  balanitis.     x  1000.         .     large  numbers,  as  was  first  shown 
by  Buschke   and  Fischer,  and   by 

Levaditi.  In  the  pemphigoid  bullse,  in  the  blood,  in  the  in- 
ternal organs,  the  liver,  lungs,  spleen,  supra-renals,  and  even  in 
the  heart  its  detection  may  be  comparatively  easy,  owing  to  the 
large  numbers  present  (Fig.  80).  It  can  readily  be  demonstrated 


232         GONORKHOEA,    SOFT   SORE,    SYPHILIS 

in  sections  of  the  organs  by  the  method  described  on  p.  104.  In 
such  preparations  large  numbers  of  spirochaetes,  chiefly  extra- 
vascular  in  position,  can  be  seen,  and  many  may  occur  in  the 
interior  of  the  more  highly  specialised  cells,  for  example,  liver- 
cells  ;  in  many  cases  examination  has  been  made  within  so  short 
a  period  after  the  death  of  the  child  as  to  practically  exclude  the 
possibility  of  contamination  from  without.  It  also  abounds 
sometimes  on  mucous  surfaces,  e.g.  of  the  bladder  and  intestine 
in  cases  of  congenital  syphilis.  Shortly  after  the  discovery  of 
the  organism,  Metchnikoff  was  able  to  detect  it  in  the  lesions 
produced  in  monkeys  by  inoculation  with  material  derived  from 
syphilitic  sores,  and  his  observations  have  since  been  confirmed. 
Although  various  organisms  may  be  associated  with  it  in  the 
lesions  of  the  skin  or  mucous  membranes,  there  is  a  comparative 
agreement  amongst  observers  that  this  organism  occurs  alone  in 
syphilitic  lesions  where  the  entrance  of  bacteria,  etc.,  from  outside 
is  excluded.  The  high  percentage  of  cases  in  which  it  is  found 
would,  in  view  of  the  difficulty  in  detecting  it,  almost  point  to 
its  invariable  presence,  and,  as  a  matter  of  fact,  Schaudinn  in 
his  last  series  of  cases,  numbering  over  seventy,  found  it  in  all. 
In  gummata  and  other  tertiary  lesions,  however,  the  spirochaete 
has  rarely,  if  ever,  been  detected,  and  it  is  probable,  as  Schaudinn 
suggests,  that  it  has  passed  into  some  resting  condition  which 
has  not  yet  been  found.  Another  question  of  considerable 
importance  is,  as  to  whether  this  organism  has  been  found  in 
other  conditions.  Observations  show  that  in  various  conditions, 
such  as  ulcerated  carcinomata,  balanitis,  etc.,  spirochsetes  are  of 
comparatively  common  occurrence.  There  is  no  doubt  what- 
ever that  the  great  majority  of  these  are  readily  distinguishable 
by  their  appearance  from  the  spirochaete  pallida,  but  others 
resemble  it  closely.  Hoffmann,  however,  who  has  seen  many  of 
these  spirochsetes  from  other  sources,  considers  that  even  by 
their  microscopic  appearance  they  are  capable  of  being  dis- 
tinguished, though  with  considerable  difficulty.  It  must,  of 
course,  be  borne  in  mind  that  the  finding  of  an  organism  in 
non-syphilitic  lesions  with  exactly  the  same  microscopical  char- 
acters does  not  show  that  it  is  the  same  organism  as  the  spiro- 
chaete pallida.  It  cannot  be  claimed  that  the  pathological 
relation  of  this  organism  to  the  disease  is  absolutely  demon- 
strated ;  but  the  facts  stated  are  sufficient  to  form  very  strong 
presumptive  evidence  that  in  the  spirochaete  pallida  we  have 
the  true  cause  of  syphilis. 

Transmission    of   the    Disease    to    Animals.  —  Although 
various   experiments   had  previously   been   from  time  to  time 


SYPHILIS  233 

made  by  different  observers,  in  some  cases  with  reported 
successful  result,  it  is  to  the  papers  of  Metchnikoff  and  Roux 
(1903-5)  that  we  owe  most  of  our  knowledge.  These  observers 
have  carried  on  a  large  series  of  observations,  and  have  shown 
that  the  disease  can  be  transmitted  to  various  species  of  monkeys. 
Of  these  the  anthropoid  apes  are  most  susceptible,  the  chimpanzee 
being  the  most  suitable  for  experimental  purposes.  Their 
results  have  been  confirmed  by  Lassar,  Neisser,  Kraus,  and 
others.  Inoculations  made  by  scarification  resulted  in  the 
production  of  typical  primary  lesions  in  all  of  more  than  twenty 
animals  used.  The  primary  lesion  is  in  the  form  of  an  indurated 
papule  or  of  papules,  in  every  respect  resembling  the  human 
lesion.  Along  with  this  there  is  a  marked  enlargement  and 
induration  of  the  corresponding  lymphatic  glands.  The  primary 
lesion  appeared  on  an  average  about  thirty  days  after  inocula- 
tion, and  secondary  symptoms  appeared  in  rather  more  than 
half  of  the  cases  after  a  further  period  of  rather  longer  duration. 
These  were  of  the  nature  of  squamous  papules  on  the  skin, 
mucous  patches  in  the  mouth,  and  sometimes  palmar  psoriasis. 
As  a  rule,  the  secondary  manifestations  were  of  a  somewhat 
mild  degree,  and  in  no  instance  up  to  the  present  has  any 
tertiary  lesion  been  observed.  By  inoculation  from  the  secondary 
lesions,  the  primary  manifestations  with  their  typical  characters 
have  been  reproduced.  The  orang-outang  has  been  found  to  be 
less .  susceptible,  whilst  Roux's  experiments  on  the  gorilla  have 
been  too  few  to  admit  of  any  conclusion.  The  disease  may  also 
be  produced  in  baboons  and  macaques  (macacus  sinicus  is  one  of 
the  most  susceptible),  but  these  animals  are  less  susceptible. 
In  the  case  of  many  of  them  no  result  follows,  and  when  a  lesion 
is  produced  it  is  only  of  the  nature  of  a  primary  papule, 
secondary  manifestations  never  appearing.  There  is  thus  no 
doubt  that  the  disease  may  be  produced  in  apes,  and,  to  speak 
generally,  the  severity  of  the  affection  increases  according  to  the 
nearness  of  the  relationship  of  the  animal  to  the  human 
subject. 

The  production  of  the  disease,  experimentally,  has  supplied 
us  with  some  further  facts  regarding  the  nature  of  the  virus. 
It  has  been  shown  repeatedly  that  the  passage  of  fluid  con- 
taining the  virus  through  a  Berkefeld  filter  deprives  it  completely 
of  its  infectivity.  In  other  words,  the  virus  does  not  belong  to 
the  ultra-microscopic  group  of  organisms.  The  virus  is  also 
readily  destroyed  by  heat,  a  temperature  of  51°  C.  being 
fatal.  With  regard  to  the  production  of  immunity,  very  little 
of  a  satisfactory  nature  has  so  far  been  established.  It  has  been 


234         GONORKHCEA,    SOFT   SORE,    SYPHILIS 

found  that  the  virus  from  a  macaque  monkey  produces  a  less 
severe  disease  in  the  chimpanzee  than  the  virus  from  the  human 
subject,  inasmuch  as  secondary  lesions  do  not  follow  ;  the  virus 
would  thus  appear  to  have  undergone  a  certain  amount  of 
attenuation  in  the  tissues  of  that  monkey.  Recently  corneal 
ulcers  in  rabbits  have  been  produced  by  Bertarelli  and  by  Hoff- 
mann by  inoculation  with  syphilitic  material ;  they  appear  after 
a  long  period  of  incubation,  and  the  spirochaete  can  be  demon- 
strated in  the  lesions.  The  effects  of  injecting  emulsions  of 
tertiary  lesions  or  of  serum  from  syphilitic  patients,  at  the  time 
of  inoculation  with  the  virus,  appear  to  be  practically  nil; 
so  also  the  employment  of  the  virus  rendered  inactive  by  heating 
has  apparently  no  influence  in  acting  as  a  vaccine.  There  is 
some  evidence  that  the  serum  from  a  patient  suffering  from  the 
disease  when  mixed  with  the  virus  before  inoculation  modifies 
the  disease  to  a  certain  extent,  but  further  evidence  on  this 
point  is  necessary.  As  mentioned  above,  the  spirochsete  pallida 
has  been  found  in  the  lesions  in  monkeys,  Metchnikoff  and 
Roux  obtaining  positive  results  in  more  than  75  per  cent  of  the 
cases,  and  it  is  to  be  noted  that  here  also  the  organism  has  been 
found  deep  in  the  substance  of  the  papules,  unaccompanied  by 
any  other  organisms.  Hoffmann  failed  to  find  any  spirochaetes 
in  monkeys  which  had  not  been  inoculated  with  syphilitic 
material.  This  observer  produced  a  lesion  on  the  upper  eyelid 
of  a  macacus  by  inoculation  with  the  blood  of  a  man  who  had 
suffered  from  the  disease  for  six  months,  and  a  papule  appeared 
which  contained  spirochaetes.  This  result  is  in  conformity  with 
that  given  by  microscopic  examination,  and  shows  that  the 
organism  is  sometimes  present  in  the  circulating  blood  in  severe 
cases  of  the  disease,  and  that  the  blood  is  accordingly  infective. 
Castellani  has  described  in  yaws  or  framboesia  the  occurrence 
of  a  spirochsete  closely  resembling  the  spirochsete  pallida  in 
appearance,  and  to  this  organism  he  has  given  the  name  spiro- 
chcete  pertenuis.  He  has  found  it  not  only  in  the  skin  lesions 
but  also  in  the  spleen  and  lymphatic  glands  of  patients  suffering 
from  the  disease.  He  has  produced  the  disease  in  monkeys  by 
direct  inoculation  and  has  found  the  spirochaete  in  the  resulting 
lesions.  He  finds  that  the  immunity  reactions  of  the  two  organ- 
isms— spirochsete  pallida  and  spirochaete  pertenuis — are  quite 
distinct ;  hence  we  have  probably  to  deal  with  two  distinct  species. 


CHAPTER   IX. 

TUBERCULOSIS. 

THE  cause  of  tubercle  was  proved  by  Koch  in  1882  to  be  the 
organism  now  universally  known  as  the  tubercle  bacillus. 
Probably  no  other  single  discovery  has  had  a  more  important 
effect  on  medical  science  and  pathology  than  this.  It  has  not 
only  shown  what  is  the  real  cause  of  the  disease,  but  has  also 
supplied  infallible  methods  for  determining  what  are  tubercular 
lesions  and  what  are  not,  and  has  also  given  the  means  of 
studying  the  modes  and  paths  of  infection.  A  definite  answer 
has  in  this  way  been  supplied  to  many  questions  which  were 
previously  the  subject  of  endless  discussion. 

Historical. — By  the  work  of  Armanni  and  of  Cohnheim  and  Salomonsen 
(1870-80)  it  had  been  demonstrated  that  tubercle  was  an  infective  disease. 
The  latter  observers  found  on  inoculation  of  the  anterior  chamber  of  the 
eye  of  rabbits  with  tubercular  material  that  in  many  cases  the  results  of 
irritation  soon  disappeared,  but  that  after  a  period  of  incubation,  usually 
about  twenty-five  days,  small  tubercular  nodules  appeared  in  the  iris  ; 
afterwards  the  disease  gradually  spread,  leading  to  a  tubercular  disorgan- 
isation of  the  globe  of  the  eye.  Later  still,  the  lymphatic  glands  became 
involved,  and  finally  the  animal  died  of  acute  tuberculosis.  The  question 
remained  as  to  the  nature  of  the  virus,  the  specific  character  of  which 
was  thus  established,  and  this  question  was  answered  by  the  work 
of  Koch. 

The  announcement  of  the  discovery  of  the  tubercle  bacillus  was  made 
by  Koch  in  March  1882,  and  a  full  account  of  his  researches  appeared  in 
1884  (Mitth.  a.  d.  K.  Gsndhtsamte.,  Berlin).  Koch's  work  on  this  subject 
will  remain  as  a  classical  masterpiece  of  bacteriological  research,  both  on 
account  of  the  great  difficulties  which  he  successfully  overcame  and  the 
completeness  with  which  he  demonstrated  the  relations  of  the  organism 
to  the  disease.  The  two  chief  difficulties  were,  first,  the  demonstration 
of  the  bacilli  in  the  tissues,  and,  secondly,  the  cultivation  of  the  organism 
outside  the  body.  For,  with  regard  to  the  first,  the  tubercle  bacillus 
cannot  be  demonstrated  by  a  simple  watery  solution  of  a  basic  aniline 
dye,  and  it  was  only  after  prolonged  staining  for  twenty-four  hours,  with 
a  solution  of  methylene-blue  with  caustic  potash  added,  that  he  was 
able  to  reveal  the  presence  of  the  organism.  Then,  in  the  second  place, 

235 


236  TUBERCULOSIS 

all  attempts  to  cultivate  it  on  the  ordinary  media  failed,  and  he  only 
succeeded  in  obtaining  growth  on  solidified  blood  serum,  the  method  of 
preparing  which  he  himself  devised,  inoculations  being  made  on  this 
medium  from  the  organs  of  animals  artificially  rendered  tubercular. 
The  fact  that  growth  did  not  appear  till  the  tenth  day  at  the  earliest, 
might  easily  have  led  to  the  hasty  conclusion  that  no  growth  took  place. 
All  difficulties  were,  however,  successfully  overcome.  He  cultivated  the 
organism  by  the  above  method  from  a  great  variety  of  sources,  and  by 
a  large  series  of  inoculation  experiments  on  various  animals,  performed 
by  different  methods,  he  conclusively  proved  that  bacilli  from  these 
different  sources  produced  the  same  tubercular  lesions  and  were  really  of 
the  same  species.  His  work  was  the  means  of  showing  conclusively  that 
such  conditions  as  lupus,  "  wrhite  swelling"  of  joints,  scrofulous  disease 
of  glands,  etc. ,  are  really  tubercular  in  nature. 

Tuberculosis  in  Animals. — Tuberculosis  is  not  only  the  most 
widely  spread  of  all  diseases  affecting  the  human  subject,  and 
produces  a  mortality  greater  than  any  other,  but  there  is  probably 
no  other  disease  which  affects  the  domestic  animals  so  widely. 
We  need  not  here  describe  in  detail  the  various  tubercular  lesions 
in  the  human  subject,  but  some  facts  regarding  the  disease  in 
the  lower  animals  may  be  given,  as  this  subject  is  of  great  im- 
portance in  relation  to  the  infection  of  the  human  subject. 

Amongst  the  domestic  animals  the  disease  is  commonest  in  cattle 
(bovine  tuberculosis),  in  which  animals  the  lesions  are  very  various,  both 
in  character  and  distribution.  In  most  cases  the  lungs  are  affected,  and 
contain  numerous  rounded  nodules,  many  being  of  considerable  size  ; 
these  may  be  softened  in  the  centre,  but  are  usually  of  pretty  firm 
consistence  and  may  be  calcified.  There  may  be  in  addition  caseous 
pneumonia,  and  also  small  tubercular  granulations.  Along  with  these 
changes  in  the  lungs,  the  pleurae  are  also  often  affected,  and  show 
numerous  nodules,  some  of  which  may  be  of  large  size,  firm  and  pedun- 
culated,  the  condition  being  known  in  Germany  as  Perlsucht,  in  France 
as  pommeliere.  Lesions  similar  to  the  last  may  be  chiefly  confined  to 
the  peritoneum  and  pleurae.  In  other  cases,  again,  the  abdominal  organs 
are  principally  involved.  The  udder  becomes  affected  in  a  certain  pro- 
portion of  cases  of  tuberculosis  in  cows — in  3  per  cent  according  to  Bang 
— but  primary  affection  of  this  gland  is  very  rare.  Tuberculosis  is  also 
a  comparatively  common  disease  in  pigs,  in  which  animals  it  in  many 
cases  affects  the  abdominal  organs,  in  other  cases  produces  a  sort  of 
caseous  pneumonia,  and  sometimes  is  met  with  as  a  chronic  disease  of 
the  lymphatic  glands,  the  so-called  "scrofula1"  of  pigs.  Tubercular 
lesions  in  the  muscles  are  less  rare  in  pigs  than  in  most  other  animals. 
In  the  horse  the  abdominal  organs  are  usually  the  primary  seat  of  the 
disease,  the  spleen  being  often  enormously  enlarged  and  crowded  with 
nodules  of  various  shapes  and  sizes  ;  sometimes,  however,  the  primary 
lesions  are  pulmonary.  In  sheep  and  goats  tuberculosis  is  of  rare 
occurrence,  especially  in  the  former  animals.  It  may  occur  spontaneously 
in  dogs,  cats,  and  in  the  large  carnivora.  It  is  also  sometimes  met  with 
in  monkeys  in  confinement,  and  leads  to  a  very  rapid  and  widespread 
affection  in  these  animals,  the  nodules  having  a  special  tendency  to 
soften  and  break  down  into  a  pus-like  fluid. 


THE   TUBERCLE   BACILLUS  237 

Tuberculosis  in  fowls  (avian  tuberculosis)  is  a  common  and  very 
infectious  disease,  nearly  all  the  birds  in  a  poultry-yard  being  sometimes 
affected. 

From  these  statements  it  will  be  seen  that  the  disease  in 
animals  presents  great  variations  in  character,  and  may  differ  in 
many  respects  from  that  met  with  in  the  human  subject.  The 
relation  of  the  different  forms  of  tuberculosis  is  discussed  below. 

Tubercle  Bacillus  —  Microscopical  Characters.  —  Tubercle 
bacilli  are  minute  rods  which  usually  measure  2  '5  to  3*5  //,  in 
length,  and  '3  //,  in  thickness,  i.e.  in  proportion  to  their  length 
they  are  comparatively  thin  organisms  (Figs.  82  and  83).  Some- 
times, however,  longer 

forms,  up  to  5  /A  or  more  ^r~gr~t  i   » 

in  length,  are  met  with,  f  s 

both  in  cultures  and  in  the  \  if 

tissues.    They  are  straight  *     \    •  » 

or  slightly  curved,  and  are      jj 
of  uniform   thickness,  or     /        i  *""    \   ^v  x 
may  show  slight  swelling     /.-_  "   {}  _       . 

at  their  extremities.  When  ^ 


stained  they  appear  uni-  ^  viX  / 

formly   coloured,   or  may     \$f         •  v_  *     </ 

present  small  uncoloured  \ 

spots  along  their  course, 

with  darkly  -stained  parts  \ 

between.     In  such  a  min-  \  (^ 

ute    organism    it    is    ex-  ^-"' 

tremely  difficult  to  deter^         FlG  82.  —Tubercle  bacilli,  from  a  pure 

mine  the  exact  nature  of  culture  on  glycerin  agar. 

the  unstained  points.     Ac-         Stained  with  carbol-fuchsin.      x   1000. 

cordingly,    we    find    that 

some  observers  consider  these  to  be  spores,  while  others  find  that 

it  is  impossible  to  stain  them  by  any  means  whatever,  and  con- 

sider that  they  are  really  of  the  nature  of  vacuoles.    Against  their 

being  spores  is  also  the  fact  that  many  occur  in  one  bacillus. 

Others  again  hold  that  some  of  the  condensed  and  highly-stained 

particles  are  spores.     It  is  impossible  to  speak  definitely  on  the 

question  at  present.     We  can  only  say  that  the  younger  bacilli 

stain  uniformly,  and  that  in  the  older  forms  inequality  in  staining 

is  met  with  ;  this  latter  condition  is,  however,  not  associated  with 

greater  powers  of  resistance. 

The  bacilli  in  the  tissues  occur  scattered  irregularly  or  in 
little  masses.  They  are  usually  single,  or  two  are  attached  end 
to  end  and  often  form  in  such  a  case  an  obtuse  angle.  True 


238  TUBERCULOSIS 

chains  are  not  formed,  but  occasionally  short  filaments  are  met 
with.  In  cultures  the  bacilli  form  masses  in  which  the  rods  are 
closely  applied  to  one  another  and  arranged  in  a  more  or  less 
parallel  manner.  Tubercle  bacilli  are  quite  devoid  of  motility. 

Aberrant  Forms. — Though  such  are  the  characters  of  the 
organism  as  usually  met  with,  other  appearances  are  sometimes 
found.  In  old  cultures,  for  example,  very  much  larger  elements 


FIG.  83. — Tubercle  bacilli  in  phthisical  sputum  ;  they  are  longer  than 

is  often  the  case. 
Film  preparation,  stained  with  carbol-fuchsiu  and  rnethylene-blue.      x  1000. 

may  occur.  These  may  be  in  the  form  of  long  filaments,  some- 
times swollen  or  clubbed  at  their  extremities,  may  be  irregularly 
beaded,  and  may  even  show  the  appearance  of  branching.  Such 
forms  have  been  studied  by  Metchnikoff,  Maffucci,  Klein,  and 
others.  Their  significance  has  been  variously  interpreted,  for 
while  some  look  upon  them  as  degenerated  or  involution  forms, 
others  regard  them  as  indicating  a  special  phase  in  the  life 
history  of  the  organism,  allying  it  with  the  higher  bacteria. 
Recent  observations,  however,  go  to  establish  the  latter  view, 
and  this  is  now  generally  accepted  by  authorities.  It  has  also 


CULTIVATION   OF   TUBERCLE   BACILLUS      239 

been  found  that  under  certain  circumstances  tubercle  bacilli  in 
the  tissues  produce  a  radiating  structure  closely  similar  to  that 
of  the  actinomyces.  This  was  found  by  Babes  and  also  by 
Lubarsch  to  be  the  case  when  the  bacilli  were  injected  under 
the  dura  mater  and  directly  into  certain  solid  organs,  such  as 
the  kidneys  in  the  rabbit.  Club-like  structures  may  be  present 
at  the  periphery ;  these  are  usually  not  acid-fast,  but  they  retain 
the  stain  in  the  Weigert-Gram  method.  Similar  results  obtained 
with  other  acid-fast  bacilli  will  be  mentioned  below,  and  these 
organisms  would  appear  to  form  a  group  closely  allied  to  the 
streptothricese,  the  bacillary  parasitic  form  being  one  stage  of 
the  life  history  of  the  organism.  This  group  is  often  spoken  of 
as  the  mycobacteria. 

Staining  Reactions. — The  tubercle  bacillus  takes  up  the 
ordinary  stains  very  slowly  and  faintly,  and  for  successful  staining 
one  of  the  most  powerful  solutions  ought  to  be  employed,  e.g. 
gentian- violet  or  fuchsin,  along  with  aniline-oil  water  or  solution 
of  carbolic  acid.  Further,  such  staining  solutions  require  to  be 
applied  for  a  long  time,  or  the  staining  must  be  accelerated  by 
heat,  the  solution  being  warmed  till  steam  arises  and  the 
specimen  allowed  to  remain  in  the  hot  stain  for  two  or  three 
minutes.  One  of  the  best  and  most  convenient  methods  is  the 
Ziehl-Neelsen  method  (see  p.  100).  The  bacilli  present  this 
further  peculiarity,  however,  that  after  staining  has  taken  place 
they  resist  decolorising  by  solutions  which  readily  remove  the 
colour  from  the  tissues  and  from  other  organisms  which  may  be 
present.  Such  decolorising  agents  are  sulphuric  or  nitric  acid 
in  20  per  cent  solution.  Preparations  can  thus  be  obtained  in 
which  the  tubercle  bacilli  alone  are  coloured  by  the  stain  first 
used,  and  the  tissues  can  then  be  coloured  by  a  contrast  stain. 
Within  recent  years  certain  other  bacilli  have  been  discovered 
which  present  the  same  staining  reactions  as  tubercle  bacilli ; 
they  are  therefore  called  "  acid-fast "  (vide  infra).  The  spores 
of  many  bacilli  become  decolorised  more  readily  than  tubercle 
bacilli,  though  some  retain  the  colour  with  equal  tenacity. 

Bullock  and  Macleod,  by  treating  tubercle  bacilli  with  hot  alcohol 
and  ether,  extracted  a  wax  which  gave  the  characteristic  staining 
reactions  of  the  bacilli  themselves.  The  remains  of  the  bacilli,  further, 
when  extracted  with  caustic  potash,  yielded  a  body  which  was  probably 
a  chitin,  and  which  was  acid-fast  when  stained  for  twenty-four  hours 
with  carbol-fuchsin. 

Cultivation. — The  medium  first  used  by  Koch  was  inspissated 
blood  serum  (vide  p.  39).  If  inoculations  are  made  on  this 
medium  with  tubercular  material  free  from  other  organisms, 


240 


TUBEECULOSIS 


there  appear  in  from  ten  to  fourteen  days  minute  points  of 
growth  of  dull  whitish  colour,  rather  irregular,  and  slightly  raised 
above  the  surface  (it  is  advisable  to  plant  on  the  medium  an 
actual  piece  of  the  tubercular  tissue  and  to  fix  it  in  a  wound  of 
the  surface  of  the  serum).  Koch  compared  the  appearance  of  these 
to  that  of  small  dry  scales.  In  such  cultures  the  growths  usually 

reach  only  a  compara- 
tively small  size  and  re- 
main separate,  becoming 
confluent  only  when  many 
occur  close  together.  In 
sub  -  cultures,  however, 
growth  is  more  luxuriant 
and  may  come  to  form  a 
dull  wrinkled  film  of 
whitish  colour,  which 
may  cover  the  greater  part 
of  the  surf  ace  of  the  serum 
and  at  the  bottom  of  the 
tube  may  grow  over  the 
surface  of  the  condensa- 
tion water  on  to  the  glass 
(Fig.  84,  A).  The  growth 
is  always  of  a  dull  ap- 
pearance and  has  a  con- 
siderable degree  of  con- 
sistence, so  that  it  is  diffi- 
cult to  dissociate  a  portion 
thoroughly  in  a  drop  of 
water.  In  older  cultures 
the  growth  may  acquire 
a  slightly  brownish  or  buff 
colour.  When  the  small 
colonies  are  examined 
under  a  low  power  of  the 
microscope  they  are  seen 

to  be  extending  at  the  periphery  in  the  form  of  wavy  or  sinuous 
streaks  which  radiate  outward  and  which  have  been  compared 
to  the  flourishes  of  a  pen.  The  central  part  shows  similar 
markings  closely  interwoven.  These  streaks  are  composed  of 
masses  of  the  bacilli  arranged  in  a  more  or  less  parallel  manner. 
"  On  glycerin  agar,  which  was  first  introduced  by  Nocard  and 
Roux  as  a  medium  for  the  culture  of  the  tubercle  bacillus, 
growth  takes  place  in  sub-cultures  at  an  earlier  date  and  pro- 


A  B  C 

FIG.  84. — Cultures  of  tubercle  bacilli  on 
glycerin  agar. 

A  and  B.  Mammalian  tubercle  bacilli ;  A  is  an 
old  culture,  B  one  of  a  few  weeks'  growth. 

C.  Avian  tubercle  bacilli.  The  growth  is  whiter 
and  smoother  on  the  surface  than  the  others. 


POWERS   OF   RESISTANCE  241 

gresses  more  rapidly  than  on  serum,  but  this  medium  is  not 
suitable  for  obtaining  cultures  from  the  tissues,  inoculations 
with  tubercular  material  usually  yielding  a  negative  result.  The 
growth  has  practically  the  same  characters  as  on  serum,  but  is 
more  luxuriant.  In  glycerin  broth,  especially  when  the  layer  is 
not  deep,  tubercle  bacilli  grow  readily  in  the  form  of  little  white 
masses  which  fall  to  the  bottom  and  form  a  powdery  layer.  If, 
however,  the  growth  be  started  on  the  surface  it  spreads  super- 
ficially as  a  dull  whitish,  wrinkled  pellicle  which  may  reach  the 
walls  of  the  flask ;  this  mode  of  growth  is  specially  suitable  for 
the  production  of  tuberculin  (vide  infra).  The  culture  has  a 
peculiar  fruity  and  not  unpleasant  odour.  On  ordinary  agar 
and  on  gelatin  media  no  growth  takes  place. 

It  was  at  one  time  believed  that  the  tubercle  bacillus  would  only  grow 
on  media  containing  animal  fluids,  but  of  late  years  it  has  been  found  that 
growth  takes  place  also  on  a  purely  vegetable  medium,  as  was  first  shown 
by  Pawlowsky  in  the  case  of  potatoes.  Sander  has  shown  that  the 
bacillus  grows  readily  on  potato,  carrot,  macaroni,  and  on  infusion  of 
these  substances,  especially  when  glycerin  is  added.  He  also  found 
that  cultures  from  tubercular  lesions  could  be  obtained  on  glycerin  potato 
(p.  46). 

The  optimum  temperature  for  growth  is  37°  to  38°  C. 
Growth  ceases  above  42°  and  usually  below  28°,  but  on  long- 
continued  cultivation  outside  the  body  and  in  special  circum- 
stances, growth  may  take  place  at  a  lower  temperature,  e.g. 
Sander  found  that  growth  took  place  in  glycerin-potato  broth 
even  at  22°  to  23°  C. 

Powers  of  Eesistance. — Tubercle  bacilli  have  considerable 
powers  of  resistance  to  external  influences,  and  can  retain  their 
vitality  for  a  long  time  outside  the  body  in  various  conditions  ; 
in  fact,  in  this  respect  they  may  be  said  to  occupy  an  inter- 
mediate position  between  spores  and  spore-free  bacilli.  Dried 
phthisical  sputum  has  been  found  to  contain  still  virulent  bacilli 
(or  their  spores)  after  two  months,  and  similar  results  are  obtained 
when  the  bacilli  are  kept  in  distilled  water  for  several  weeks. 
So  also  they  resist  for  a  long  time  the  action  of  putrefaction, 
which  is  rapidly  fatal  to  many  pathogenic  organisms.  Sputum 
has  been  found  to  contain  living  tubercle  bacilli  even  after  being 
allowed  to  putrefy  for  several  weeks  (Fraenkel,  Baumgarten),  and 
the  bacilli  have  been  found  to  be  alive  in  tubercular  organs  which 
have  been  buried  in  the  ground  for  a  similar  period.  They  are 
not  killed  by  being  exposed  to  the  action  of  the  gastric  juice  for 
six  hours,  or  to  a  temperature  of  -  3°  C.  for  three  hours,  even 
when  this  is  repeated  several  times.  It  has  been  found  that 
16 


242  TUBERCULOSIS 

when  completely  dried  they  can  resist  a  temperature  of  100°  C. 
for  an  hour,  but,  on  the  other  hand,  exposure  in  the  moist 
condition  to  70°  C.  for  the  same  time  is  usually  fatal.  It  may 
be  stated  that  raising  the  temperature  to  100°  C.  kills  the  bacilli 
in  fluids  and  in  tissues,  but  in  the  case  of  large  masses  of  tissue 
care  must  be  taken  that  this  temperature  is  reached  throughout. 
They  are  killed  in  less  than  a  minute  by  exposure  to  5  per  cent 
carbolic  acid,  and  both  Koch  and  Straus  found  that  they  are 
rapidly  killed  by  being  exposed  to  the  action  of  direct  sunlight. 

Action  on  the  Tissues. — The  local  lesion  produced  by  the 
tubercle  bacillus  is  the  well-known  tubercle  nodule,  the 
structure  of  which  varies  in  different  situations  and  according  to 
the  intensity  of  the  action  of  the  bacilli.  After  the  bacilli  gain 
entrance  to  a  connective  tissue  such  as  that  of  the  iris,  their 
first  action  appears  to  be  on  the  connective-tissue  cells,  which 
become  somewhat  swollen  and  undergo  mitotic  division,  the 
resulting  cells  being  distinguishable  by  their  large  size  and  pale 
nuclei — the  so-called  epithelioid  cells.  These  proiif erative  changes 
may  be  well  seen  on  the  fifth  day  after  inoculation  or  even 
earlier.  A  small  focus  of  proliferated  cells  is  thus  formed  in  the 
neighbourhood  of  the  bacilli  and  about  the  same  time  numbers 
of  leucocytes — chiefly  lymphocytes — begin  to  appear  at  the 
periphery  and  gradually  become  more  numerous.  Soon,  however, 
the  action  of  the  bacilli  as  cell-poisons  comes  into  prominence. 
The  epithelioid  cells  become  swollen  and  somewhat  hyaline,  their 
outlines  become  indistinct,  whilst  their  nucleus  stains  faintly, 
and  ultimately  loses  the  power  of  staining.  The  cells  in  the 
centre,  thus  altered,  gradually  become  fused  into  a  homogeneous 
substance  and  this  afterwards  becomes  somewhat  granular  in 
appearance.  If  the  central  necrosis  does  not  take  place  quickly, 
then  giant-cell  formation  may  occur  in  the  centre  of  the  follicle, 
this  constituting  one  of  the  characteristic  features  of  the  tuber- 
cular lesion ;  or  after  the  occurrence  of  caseation  giant-cells  may 
be  formed  in  the  cellular  tissue  around.  The  centre  of  a  giant- 
cell  often  shows  signs  of  degeneration,  such  as  hyaline  change 
and  vacuolation,  or  it  may  be  more  granular  than  the  rest  of  the 
cell. 

Though  there  has  been  a  considerable  amount  of  discussion 
as  to  the  mode  of  origin  of  the  giant-cells,  we  think  there  can 
be  little  doubt  that  in  most  cases  they  result  from  enlargement 
of  single  epithelioid  cells,  the  nucleus  of  which  undergoes  pro- 
liferation without  the  protoplasm  dividing.  These  epithelioid 
cells  may  sometimes  be  the  lining  cells  of  capillaries.  Some  con- 
sider that  the  giant-cells  result  from  a  fusion  of  the  epithelioid 


ACTION   ON   THE   TISSUES  243 

cells ;  but,  though  there  are  occasionally  appearances  which 
indicate  such  a  mode  of  formation,  it  cannot  be  regarded  as  of 
common  occurrence.  In  some  cases  of  acute  tuberculosis,  when 
the  bacilli  become  lodged  in  a  capillary  the  endothelial  cells  of 
its  wall  may  proliferate,  and  thus  a  ring  of  nuclei  may  be  seen 
round  a  small  central  thrombus.  Such  an  occurrence  gives  rise 
to  an  appearance  closely  resembling  a  typical  giant -cell. 
According  to  the  view  here  stated,  both  the  epithelioid  and  the 
giant-cells  are  of  connective  tissue  origin ;  and  we  can  see  no 
sufficient  evidence  for  the  view  held  by  some  observers,  chiefly 
of  the  French  school,  that  they  are  formed  from  leucocytes 
which  have  emigrated  from  the  capillaries. 

There  can  be  no  doubt  that  the  cell  necrosis  and  subsequent 
caseation  depend  upon  the  products  of  the  bacilli,  and  are  not  due 
to  the  fact  that  the  tubercle  nodule  is  non- vascular.  This  non- 
vascularity  itself  is  to  be  explained  by  the  circumstance  that 
young  capillaries  cannot  grow  into  a  part  where  tubercle  bacilli 
are  active,  and  that  the  already  existing  capillaries  become 
thrombosed,  owing  to  the  action  of  the  bacillary  products  on 
their  walls,  and  ultimately  disappear.  At  the  periphery  of 
tubercular  lesions  there  may  be  considerable  vascularity  and  new 
formation  of  capillaries. 

The  general  symptoms  of  tuberculosis — pyrexia,  perspiration, 
wasting,  etc.,  are  to  be  ascribed  to  the  absorption  and  distribution 
throughout  the  system  of  the  toxic  products  of  the  bacilli ;  in 
the  case  of  phthisical  cavities  and  like  conditions  where  other 
bacteria  are  present,  the  toxins  of  the  latter  also  play  an  im- 
portant part.  The  occurrence  of  waxy  change  in  the  organs  is 
believed  by  some  to  be  chiefly  due  to  the  products  of  other, 
especially  pyogenic,  organisms,  secondarily  present  in  the  tuber- 
cular lesions.  This  matter,  however,  requires  further  elucidation. 

Presence  and  Distribution  of  the  Bacilli. — A  few  facts  may  be 
stated  regarding  the  presence  of  bacilli,  and  the  numbers  in 
which  they  are  likely  to  be  found  in  tubercular  lesions.  On  the 
one  hand,  they  may  be  very  few  in  number  and  difficult  to  find, 
and  on  the  other  hand,  they  may  be  present  in  very  large 
numbers,  sometimes  forming  masses  which  are  easily  visible  under 
the  low  power  of  the  microscope. 

They  are  usually  very  few  in  number  in  chronic  lesions, 
whether  these  are  tubercle  nodules  with  much  connective  tissue 
formation  or  old  caseous  collections.  In  caseous  material  one 
can  sometimes  see  a  few  bacilli  faintly  stained,  along  with  very 
minute  unequally  stained  granular  points,  some  of  which  may 
possibly  be  spores  of  the  bacilli.  Whether  they  are  spores  or 


244 


TUBERCULOSIS 


not,  the  important  fact  has  been  established  that  tubercular 
material  in  which  no  bacilli  can  be  found  microscopically,  may 
be  proved,  on  experimental  inoculation  into  animals,  to  be  still 
virulent.  In  such  cases  the  bacilli  may  be  present  in  numbers  so 
small  as  to  escape  observation,  or  it  may  be  that  their  spores  only 
are  present.  In  subacute  lesions,  with  well-formed  tubercle 
follicles  and  little  caseation,  the  bacilli  are  generally  scanty. 


FIG.  85. — Tubercle  bacilli  in  section  of  human  lung  in  acute  phthisis.  The 
bacilli  are  seen  lying  singly,  and  also  in  large  masses  to  left  of  field.  The 
pale  background  is  formed  by  caseous  material. 

Stained  with  carbol-fuchsin  and  Bismarck -brown,      x  1000. 

They  are  most  numerous  in  acute  lesions,  especially  where 
caseation  is  rapidly  spreading,  for  example,  in  such  conditions  as 
caseous  catarrhal  pneumonia  (Fig.  85),  acute  tuberculosis  of  the 
spleen  in  children,  which  is  often  attended  with  a  good  deal  of 
rapid  caseous  change,  etc.  In  acute  miliary  tuberculosis  a  few 
bacilli  can  generally  be  found  in  the  centre  of  the  follicles ;  but 
here  they  are  often  much  more  scanty  than  one  would  expect. 
The  tubercle  bacillus  is  one  which  not  only  has  comparatively 
slow  growth,  but  retains  its  form  and  staining  power  for  a  much 


ACTION   ON   THE   TISSUES 


245 


longer  period 
extra-cellular 
giant-cells,  in 
manner  at  the 
in  leucocytes. 
The  above 
in  the  human 


than  most  organisms.  As  a  rule  the  bacilli  are 
in  position.  Occasionally  they  occur  within  the 
which  they  may  be  arranged  in  a  somewhat  radiate 

periphery,  occasionally  also  in  epithelioid  cells  and 

statements,  however,  apply  only  to  tuberculosis 
subject,  and  even  in  this  case  there  are  exceptions. 


FIG.  86. — Tubercle  bacilli  in  giant-cells,  showing  the  radiate  arrangement 

at  the  periphery  of  the  cells.     Section  of  tubercular  udder  of  cow. 

Stained  with  carbol-fuchsin  and  Bismarck-brown.      x  1000. 

In  the  ox,  on  the  other  hand,  the  presence  of  tubercle  bacilli 
within  giant-cells  is  a  very  common  occurrence ;  and  it  is 
also  common  to  find  them  in  considerable  numbers  scattered 
irregularly  throughout  the  cellular  connective  tissue  of  the  lesions, 
even  when  there  is  little,  or  no  caseation  present  (Fig.  86). 

In  tuberculosis  in  the  horse  and  in  avian  tuberculosis  the 
numbers  of  bacilli  may  be  enormous,  even  in  lesions  which  are 
not  specially  acute ;  and  considerable  variation  both  in  their 
number  and  in  their  site  is  met  with  in  tuberculosis  of  other 
animals. 


246 


TUBERCULOSIS 


In  discharges  from  tubercular  lesions  which  are  breaking 
down,  tubercle  bacilli  are  usually  to  be  found.  In  the  sputum  of 
phthisical  patients  their  presence  can  be  demonstrated  almost 
invariably  at  some  period,  and  sometimes  their  numbers  are  very 
large  (for  method  of  staining  see  p.  101).  Several  examinations 
may,  however,  require  to  be  made ;  this  should  always  be  done 
before  any  conclusion  as  to  the  non- tubercular  nature  of  a  case  is 

come  to.  In  cases  of  genito- 
urinary tuberculosis  they 
are  usually  present  in  the 
urine ;  but  as  they  are 
much  diluted  it  is  difficult 
to  find  them  unless  a  de- 
posit is  obtained  by  means 
of  the  centrifuge.  This 
deposit  is  examined  in  the 
same  way  as  the  sputum. 
The  bacilli  often  occur  in 
little  clumps,  as  shown  in 
Fig.  87.  In  tubercular 
ulceration  of  the  intestine 
their  presence  in  the  faeces 
may  be  demonstrated,  as 
FIG.  87. — Tubercle  bacilli  in  urine  ;  showing  was  first  shown  by  Koch  \ 
one  of  the  characteristic  clumps,  in  which  but  jn  ^js  cage  their  dis- 
they  often  occur.  •  -,-,  f  *•. ,-, 

Stained  with  carbol-fuchsin  and  methylene-    COVerV  1S  USUallv  °f  .llttle 
blue,     x  1000.  importance,  as  the  intes- 

tinal  lesions,  as   a   rule, 

occur  only  in  advanced  stages  when  diagnosis  is  no  longer  a 
matter  of  doubt. 

Experimental  Inoculation. — Tuberculosis  can  be  artificially 
produced  in  animals  by  infection  in  a  great  many  different  ways 
— by  injection  of  the  bacilli  into  the  subcutaneous  tissue,  into 
the  peritoneum,  into  the  anterior  chamber  of  the  eye,  into  the 
veins ;  by  feeding  the  animals  with  the  bacilli ;  and,  lastly,  by 
making  them  inhale  the  bacilli  suspended  in  the  air. 

The  exact  result,  of  course,  varies  in  different  animals  and 
according  to  the  method  of  inoculation,  but  we  may  state 
generally  that  when  introduced  into  the  tissues  of  a  susceptible 
animal,  the  bacilli  produce  locally  the  lesions  above  described, 
terminating  in  caseation  ;  that  there  occurs  a  tubercular  affection 
of  the  neighbouring  lymphatic  glands,  and  that  lastly  there 
may  be  a  rapid  extension  of  the  bacilli  to  other  organs  by  the 
blood  stream  and  the  production  of  general  tuberculosis.  Of 


INOCULATION  247 

the  animals  generally  used  for  the  purpose,  the  guinea-pig   is 
most  susceptible. 

When  a  guinea-pig  is  inoculated  subcutaneously  with  tubercle 
bacilli  from  a  culture,  or  with  material  containing  them,  such  as 
phthisical  sputum,  a  local  swelling  gradually  forms  which  is 
usually  well  marked  about  the  tenth  day.  This  swelling  becomes 
softened  and  caseous,  and  may  break  down,  leading  to  the 
formation  of  an  irregularly  ulcerated  area  with  caseous  lining. 
The  lymphatic  glands  in  relation  to  the  parts  can  generally  be 
found  to  be  enlarged  and  of  somewhat  firm  consistence,  about 
the  end  of  the  second  or  third  week.  Later,  in  them  also  caseous 
change  occurs,  and  a  similar  condition  may  spread  to  other 
groups  of  glands  in  turn,  passing  also  to  those  on  the  other  side 
of  the  body.  During  the  occurrence  of  these  changes,  the  animal 
loses  weight,  gradually  becomes  cachectic,  and  ultimately  dies, 
sometimes  within  six  weeks,  sometimes  not  for  two  or  three 
months.  Post  mortem,  in  addition  to  the  local  and  glandular 
changes,  an  acute  tuberculosis  is  usually  present,  the  spleen 
being  specially  affected.  This  organ  is  swollen,  and  is  studded 
throughout  by  numerous  tubercle  nodules,  which  may  be  minute 
and  grey,  or  larger  and  of  a  yellowish  tint.  If  death  has  been 
long  delayed,  calcification  may  have  occurred  in  some  of  the 
nodules.  Tubercle  nodules,  though  rather  less  numerous,  are 
also  present  in  the  liver  and  in  the  lungs,  the  nodules  in  the 
latter  organs  being  usually  of  smaller  size  though  occasionally  in 
large  numbers.  The  extent  of  the  general  infection  varies ; 
sometimes  the  chronic  glandular  changes  constitute  the  out- 
standing feature. 

Intraperitoneal  injection  of  pure  cultures  produces  a  local  lesion  in  the 
form  of  an  extensive  tubercular  infiltration  and  thickening  of  the 
omentum,  sometimes  attended  with  acute  tubercles  all  over  the 
peritoneum,  There  is  a  caseous  enlargement  of  the  retroperitoneal  and 
other  lymphatic  glands,  and  later  there  may  be  a  general  tuberculosis. 
Intravenous  injection  produces  a  typical  acute  tuberculosis,  the  nodules 
being  usually  more  numerous  and  of  smaller  size,  while  death  follows 
more  rapidly,  the  larger  the  numbers  of  bacilli  injected.  Guinea-pigs, 
when  fed  with  tubercle  bacilli,  or  with  sputum  or  portions  of  tissue 
containing  them,  readily  contract  an  intestinal  form  of  tuberculosis, 
lesions  being  present  in  the  lymphoid  tissue  of  the  intestines,  in  the 
mesenteric  glands,  and  later  in  the  internal  organs. 

Rabbits  are  less  susceptible  than  guinea-pigs,  and  in  them  the  effects 
of  subcutaneous  inoculation  are  very  variable  ;  sometimes  the  lesions 
remain  local,  sometimes  a  general  tuberculosis  is  set  up.  Otherwise  the 
reactions  are  much  of  the  same  nature.  Dogs  are  much  more  highly 
resistant,  but  tuberculosis  can  be  produced  in  them  by  intraperitoneal 
injection  of  pure  cultures  (Koch),  or  by  intravenous  injection  (Maffucci). 
In  the  latter  case  there  results  an  extensive  eruption  of  minute  miliary 


248  TUBERCULOSIS 

tubercles.      Tuberculosis  can  also  be   easily   produced  in  susceptible 
animals  by  making  them  inhale  the  bacilli. 

Varieties  of  Tuberculosis.  1 .  Human  and  Bovine  Tuberculosis. 
— Up  till  recent  years  it  was  generally  accepted  that  all 
mammalian  tuberculosis  was  due  to  the  same  organism,  and 
in  particular  that  tuberculosis  could  be  transmitted  from  the 
ox  to  the  human  subject.  The  matter  became  one  of  special 
interest  owing  to  Koch's  address  at  the  Tuberculosis  Congress 
in  1901,  in  which  he  stated  his  conclusion  that  human  and 
bovine  tuberculosis  are  practically  distinct,  and  that  if  a 
susceptibility  of  the  human  subject  to  the  latter  really  exists, 
infection  is  of  very  rare  occurrence, — so  rare  that  it  is  not 
necessary  to  take  any  measures  against  it.  Previously  to  this, 
Theobald  Smith  had  pointed  out  differences  between  mammalian 
and  bovine  tubercle  bacilli,  the  most  striking  being  that  the  latter 
possess  a  much  higher  virulence  to  the  guinea-pig,  rabbit,  and  other 
animals,  and  in  particular  that  human  tubercle  bacilli,  on 
inoculation  into  oxen,  produce  either  no  disease  or  only  local 
lesions  without  any  dissemination.  Koch's  conclusions  were  based 
chiefly  on  the  result  of  his  inoculations  of  the  bovine  species 
with  human  tubercle  bacilli,  the  result  being  confirmatory  of 
Smith's,  and,  secondly,  on  the  supposition  that  infection  jof  the 
human  subject  through  the  intestine  is  of  very  rare  occurrence. 

Since  the  time  of  Koch's  communication  an  enormous  amount 
of  work  has  been  done  on  this  subject,  and  Commissions  of 
inquiry  have  been  appointed  in  various  countries.  We  may 
summarise  the  chief  facts  which  have  been  established. 
Practically  all  observers  are  agreed  that  there  are  two  chief 
types  of  tubercle  bacilli  which  differ  both  in  their  cultural 
characters  and  in  their  virulence — a  bovine  type  and  a  human 
type.  The  bacilli  of  the  bovine  type  when  cultivated  are  shorter 
and  thicker  and  more  regular  in  size;  whilst  their  growth  on 
various  culture  media  is  scantier  than  that  of  the  human  type. 
From  the  latter  character  the  British  Royal  Commission  have 
applied  the  term  dysgonic  to  the  bovine  and  eugonic  to  the 
human  type.  As  already  stated  there  is  also  a  great  difference 
in  virulence  towards  the  lower  animals,  the  bacillus  from  the  ox 
having  a  much  higher  virulence.  This  organism  when  injected 
in  suitable  quantities  into  the  ox  produces  a  local  tubercular 
lesion,  which  is  usually  followed  by  a  generalised  and  fatal 
tuberculosis ;  whereas  injection  of  human  tubercle  bacilli  pro- 
duces no  more  than  a  local  lesion,  which  undergoes  retrogression. 
(In  certain  experiments,  e.g.  those  of  Delepine,  Hamilton  and 


VARIETIES   OF   TUBERCULOSIS  249 

Young,  general  tuberculosis  has  been  produced  by  tubercle 
bacilli  from  the  human  subject,  but  these  results  are  exceptional). 
Corresponding  differences  come  out  in  the  case  of  the  rabbit ;  in 
fact,  intravenous  injection  of  suitable  quantities  in  this  animal  is 
the  readiest  method  of  distinguishing  the  two  types — an  acute 
tuberculosis  resulting  with  the  bovine,  but  not  with  the  human 
type.  In  guinea-pigs  and  monkeys  a  generalised  tuberculosis  may 
result  from  subcutaneous  injection  of  bacilli  of  the  human  type, 
but  in  this  case  also  the  difference  in  favour  of  the  greater  viru- 
lence of  the  bovine  type  is  made  out.  With  regard  to  the  dis- 
tribution of  the  two  types  of  organisms,  it  may  be  stated  that  so 
far  as  we  know  the  bacillus  obtained  from  bovine  tuberculosis  is 
always  of  the  bovine  type,  and  the  same  may  be  said  to  be  true 
of  tuberculosis  in  pigs ;  in  fact  this  seems  to  be  the  prevalent 
organism  in  animal  tuberculosis.  In  human  tuberculosis  the 
bacilli  in  a  large  majority  of  the  cases  are  of  the  human  type ; 
but  on  the  other  hand,  in  a  certain  proportion  bacilli  of  the 
bovine  type  are  present,  the  bacilli  when  cultivated  being 
indistinguishable  by  any  means  at  our  disposal  from  those 
obtained  from  bovine  tuberculosis.  The  Royal  Commission 
found  the  bovine  type  in  14  out  of  60  cases  of  human 
tuberculosis  —  a  somewhat  higher  proportion  than  has  been 
obtained  by  most  other  investigators — and  in  all  of  these, 
with  one  exception,  the  bacilli  were  obtained  either  from  caseous 
cervical  glands,  or  from  the  lesions  of  primary  abdominal  tubercu- 
losis, that  is  from  cases  where  there  was  evidence  of  infection  by 
alimentation.  It  is  also  to  be  noted  that  almost  all  the  tuber- 
cular lesions  from  which  the  bovine  type  has  been  obtained  have 
been  in  children.  The  general  result  accordingly  is  that  bovine 
tubercle  bacilli  are  present  in  a  certain  proportion  of  cases  of  tuber- 
culosis in  young  subjects,  and  that  these  are  especially  cases  where 
infection  by  the  alimentary  canal  has  occurred.  It  must  thus 
be  held  as  established  that  tuberculosis  is  transmissible  from 
the  ox  to  man,  and  that  the  milk  of  tubercular  cows  is  a  common 
vehicle  of  transmission. 

Although  most  of  the  bacilli  which  have  been  cultivated 
correspond  to  one  of  the  two  types,  as  above  described,  it  is 
also  to  be  noted  that  intermediate  varieties  are  met  with.  It 
has  also  been  found  that  the  type  characters  of  the  bacillus  are 
not  constant.  Various  observers  have  found  it  possible  to 
modify  bacilli  of  the  human  type  by  passing  them  through  the 
bodies  of  certain  animals,  e.g.  guinea-pigs,  sheep,  and  goats,  so 
that  they  acquire  the  characters  of  bovine  bacilli.  In  view 
of  these  facts  it  is  probable  that  bovine  bacilli  will  undergo 


250  TUBERCULOSIS 

corresponding  modifications  in  the  tissues  of  the  human  subject 
— what  period  of  time  is  necessary  for  such  a  change  we  cannot 
say.  It  is  thus  possible  that  the  cases  from  which  the  bovine 
type  has  been  obtained  do  not  represent  the  full  number  where 
infection  from  the  ox  has  occurred.  It  is  quite  likely  that 
although  the  bovine  bacilli  are  more  virulent  to  the  lower 
animals  than  the  human  bacilli  are,  this  does  not  also  hold  in  the 
case  of  the  human  subject.  In  fact  the  comparative  chronicity 
of  the  primary  abdominal  lesions  in  children  in  the  first  instance 
would  point  rather  to  a  low  order  of  virulence  towards  the 
human  subject.  We  may  also  add  that  there  are  cases,  notably 
those  of  Ravenel,  in  which  accidental  inoculation  of  the  human 
subject  with  bovine  tubercle  has  resulted  in  the  production  of 
tuberculosis. 

2.  Avian  Tuberculosis. — In  the  tubercular  lesions  in  birds  there 
are  found  bacilli  which  correspond  in  their  staining  reactions 
and  in  their  morphological  characters  with  those  in  mammals, 
but  differences  are  observed  in  cultures,  and  also  on  experimental 
inoculation.  These  differences  were  first  described  by  Maffucci 
and  by  Rivolta,  but  special  attention  was  drawn  to  the  subject 
by  a  paper  read  by  Koch  at  the  International  Medical  Congress  in 
1890.  Koch  stated  that  he  had  failed  to  change  the  one  variety 
of  tubercle  bacillus  into  the  other,  though  he  did  not  conclude 
therefrom  that  they  were  quite  distinct  species.  The  following 
points  of  difference  may  be  noted  : — 

On  glycerin  agar  and  on  serum,  the  growth  of  tubercle  bacilli  from 
birds  is  more  luxuriant,  has  a  moister  appearance  (Fig.  84,  C),  and, 
moreover,  takes  place  at  a  higher  temperature,  43 '5°  C.,  than  is  the 
case  with  ordinary  tubercle  bacilli.  Experimental  inoculation  brings 
out  even  more  distinct  differences.  Tubercle  bacilli  derived  from  the 
human  subject,  for  example,  when  injected  into  birds,  usually  fail  to 
produce  tuberculosis,  whilst  those  of  avian  origin  very  readily  do  so. 
Birds  are  also  very  susceptible  to  the  disease  when  fed  with  portions 
of  the  organs  of  birds  containing  tubercle  bacilli,  but  they  can  consume 
enormous  quantities  of  phthisical  sputum  without  becoming  tubercular 
(Straus,  Wurtz,  Nocard).  No  doubt,  on  the  other  hand,  there  are  cases 
on  record  in  which  the  source  of  infection  of  a  poultry-yard  has  ap- 
parently been  the  sputum  of  phthisical  patients.  Again,  tubercle 
bacilli  cultivated  from  birds  have  not  the  same  effect  on  inoculation 
of  mammals  as  ordinary  tubercle  bacilli  have.  When  guinea-pigs  are 
inoculated  subcutaneously  they  usually  resist  infection,  though  occa- 
sionally a  fatal  result  follows.  In  the  latter  case,  usually  no  tubercles 
visible  to  the  naked  eye  are  found,  but  numerous  bacilli  may  be  present 
in  internal  organs,  especially  in  the  spleen,  which  is  much  swollen. 
Further,  intravenous  injection  even  of  large  quantities  of  avian  tubercle 
bacilli,  in  the  case  of  dogs,  leads  to  no  effect,  whereas  ordinary  tubercle 
bacilli  produce  acute  tuberculosis.  [The  rabbit,  on  the  other  hand,  is 
comparatively  susceptible  to  avian  tuberculosis  (Nocard).] 


VARIETIES   OF  TUBERCULOSIS  251 

There  is,  therefore,  abundant  evidence  that  the  bacilli  derived 
from  the  two  classes  of  animals  show  important  differences,  and, 
reasoning  from  analogy,  we  might  infer  that  probably  the  human 
subject  also  would  be  little  susceptible  to  infection  from  avian 
tuberculosis.  The  question  remains,  are  these  differences  of  a 
permanent  character  ?  The  matter  seems  conclusively  settled  by 
the  experiments  of  Nocard,  in  which  mammalian  tubercle  bacilli 
have  been  made  to  acquire  all  the  characters  of  those  of  avian 
origin.  The  method  adopted  was  to  place  bacilli  from  human 
tuberculosis  in  small  collodion  sacs  (v.  p.  123)  containing  bouillon, 
and  then  to  insert  each  sac  in  the  peritoneal  cavity  of  a  fowl. 
The  sacs  were  left  in  situ  for  periods  of  from  four  to  eight  months. 
They  were  then  removed,  cultures  were  made  from  their  con- 
tents, fresh  sacs  were  inoculated  from  these  cultures  and  intro- 
duced into  other  fowls.  In  such  conditions  the  bacilli  are 
subjected  only  to  the  tissue  juices,  the  wall  of  the  sac  being 
impervious  both  to  bacilli  and  to  leucocytes,  etc.  After  one 
sojourn  of  this  kind,  and  still  more  so  after  two,  the  bacilli  are 
found  to  have  acquired  some  of  the  characters  of  avian  tubercle 
bacilli,  but  are  still  non- virulent  to  fowls.  After  the  third 
sojourn,  however,  they  have  acquired  this  property,  and  produce 
in  fowls  the  same  lesion  as  bacilli  derived  from  avian  tuber- 
culosis. It  therefore  appears  that  the  bacilli  of  avian  tuberculosis 
are  not  a  distinct  and  permanent  species,  but  a  variety  which 
has  been  modified  by  growth  in  the  tissues  of  the  bird.  Evidently 
also  there  are  degrees  of  this  modification  according  to  the 
period  of  time  during  which  the  bacilli  have  passed  from  bird  to 
bird,  as  in  some  cases  inoculation  with  tubercle  bacilli  of  avian 
origin  has  produced  ordinary  tubercle  nodules  in  guinea-pigs 
(Courmont  and  Dor).  It  is  also  interesting  to  note  that 
Rabino  witch  has  cultivated  tubercle  bacilli  of  the  mammalian 
type  from  some  cases  of  tuberculosis  in  parrots  kept  in  con- 
finement. 

3.  Tuberculosis  in  the  Fish. — Bataillon,  Dubard,  and  Terre 
cultivated  from  a  tubercle-like  disease  in  a  carp,  a  bacillus  which, 
in  staining  reaction  and  microscopic  characters,  closely  agrees 
with  the  tubercle  bacillus.  The  lesion  with  which  it  was 
associated  was  an  abundant  growth  of  granulation  tissue  in 
which  numerous  giant-cells  were  present.  It  forms,  however, 
luxuriant  growth  at  the  room  temperature,  the  growth  being 
thick  and  moist  like  that  of  avian  tubercle  bacilli  (Fig.  89,  c). 
Growth  does  not  occur  at  the  body  temperature,  though  by 
gradual  acclimatisation  a  small  amount  of  growth  has  been 
obtained  up  to  36°  C.  Furthermore,  the  organism  appears  to 


252  TUBERCULOSIS 

undergo  no  multiplication  when  injected  into  the  tissues  of 
mammals,  and  attempts  to  modify  this  characteristic  have  so 
far  been  unsuccessful.  Weber  and  Taute  have  cultivated  this 
organism  from  mud,  and  also  from  organs  of  healthy  frogs.  It 
is  thus  probably  to  be  regarded  as  a  saprophyte  which  is  only 
occasionally  associated  with  disease  in  the  fish.  According  to 
the  results  of  different  experimenters  it  is  possible  to  modify 
human  tubercle  bacilli  by  allowing  them  to  sojourn  in  the  tissues 
of  cold-blooded  animals,  e.g.  the  frog,  blind-worm,  etc.,  so  that 
they  flourish  at  lower  temperatures.  These  results  have,  how- 
ever, been  recently  called  in  question,  as  it  has  been  stated  the 
organisms  obtained  were  not  modified  tubercle  bacilli  but  other 
acid-fast  bacilli  which  may  be  found  in  the  tissues  of  normal 
cold-blooded  animals.  This  question  must  accordingly  be 
considered  still  an  open  one. 

All  the  above  facts  taken  together  indicate  that  tubercle 
bacilli  may  become  modified  in  relative  virulence  and  in  con- 
ditions of  growth  by  sojourn  in  the  tissues  of  various  animals. 
This  modification  appears  slight,  though  of  definite  character  in 
the  case  of  bovine  tuberculosis,  more  distinct  in  the  case  of 
avian  tuberculosis,  and  much  more  marked,  if  not  permanent,  in 
the  case  of  fish  tuberculosis,  that  is,  of  course,  in  their  relations 
to  the  bacilli  from  the  human  subject. 

Other  Acid-fast  Bacilli. — Within  recent  years  a  number  of 
bacilli  presenting  the  same  staining  reaction  as  the  tubercle 
bacilli  have  been  discovered.  Such  bacilli  have  a  comparatively 
wide  distribution  in  nature,  as  they  have  been  obtained  from 
various  species  of  grass,  from  butter  and  milk,  from  manure,  and 
from  the  surfaces  of  animal  bodies.  Microscopically,  they  agree 
more  or  less  closely  with  tubercle  bacilli,  though  most  of  them 
are  shorter  and  plumper ;  many  of  them  show  filamentous  and 
branching  forms  under  certain  conditions  of  culture.  Moreover, 
on  injection,  they  produce  granulation-tissue  nodules  which  may 
closely  resemble  tubercles,  although  on  the  whole  there  is  a 
greater  tendency  to  softening  and  suppuration,  and  usually  the 
lesions  are  localised  to  the  site  of  inoculation.  The  most  im- 
portant point  of  distinction  is  the  fact  that  their  multiplication 
on  artificial  media  is  much  more  rapid,  growth  usually  being 
visible  within  forty-eight  hours  and  often  within  twenty-four 
hours  at  37°  C.  Furthermore,  in  most  instances,  growth  occurs 
at  the  room  temperature.  The  general  character  of  the  cultures 
in  this  group  is  a  somewhat  irregular  layer,  often  with  wrinkled 
surface,  dry  or  moist  in  appearance,  and  varying  in  tint  from 
white  to  yellow  or  reddish  brown.  The  number  of  such 


OTHER   ACID-FAST   BACILLI 


253 


organisms  is  constantly  being  added  to,  but  the  following  may 
be  mentioned  as  examples  : — 

Moeller's  Grass  Bacilli,  I.  atid  II. — The  former  was  found  in  infusions 
of  Timothy-grass  (Phleum  pratense).  It  is  extremely  acid-fast,  morpho- 
logically resembles  the  tubercle  bacillus,  and  in  cultures  may  show  club 
formation  and  branching.  The  lesions  produced  closely  resemble 
tubercles.  The  colonies,  visible  in  thirty-six  hours,  are  scale-like  and 
of  greyish-white  colour  (Fig.  89,  a).  Moeller's  bacillus  II.  was  obtained 
from  the  dust  of  a  hay-loft.  The  colonies  at  first  are  moist  and  some- 
what tenacious,  but  afterwards  run  together,  and  are  of  a  dull  yellowish 
colour.  The  general  results  of  inoculation  resemble  those  of  grass 


FIG.  88. — Moeller's  Timothy-grass  bacillus. 

From  a  culture  on  agar. 

Stained  with  carbol-fuchsin,  and  treated  with 
20  per  cent  sulphuric  acid,      x  1000. 


FIG.  89. — Cultures  of  acid-fast  bacilli 
grown  at  room  temperature. 

(a)  Moeller's  Timothy-grass  bacillus  I. 
(6)  The  Petri-Rabinowitch  butter  bacillus, 
(c)  Bacillus  of  fish  tuberculosis. 


bacillus  I.  but  are  less  marked.  Moeller  also  obtained  a  similar  organism 
from  milk.  He  also  discovered  a  third  acid-fast  bacillus  which  he 
obtained  from  manure  and  therefore  called  the  "  Mistbacillus "  (dung 
bacillus).  This  organism  has  analogous  characters,  though  presenting 
minor  differences.  It  also  has  pathogenic  effects. 

Petri  and  Rabinowitch  independently  cultivated  an  acid-fast  bacillus 
from  butter  ("butter  bacillus")  in  which  it  occurs  with  comparative 
frequency.  The  organism  resembles  the  tubercle  bacillus,  although  it  is 
on  the  whole  shorter  and  thicker.  Its  lesions  closely  resemble  tuber- 
culosis, especially  when  injection  of  the  organism  is  made  into  the 
peritoneal  cavity  of  guinea-pigs,  along  with  butter, — the  method  usually 
adopted  in  searching  for  tubercle  bacilli  in  butter.  This  organism 
produces  pretty  rapidly  a  wrinkled  growth  (Fig.  89,  b)  not  unlike  that 
of  Moeller's  grass  bacillus  II.  Korn  has  also  obtained  other  two 
bacilli  from  butter  which  he  holds  to  be  distinct  from  one  another  and 


254 


TUBERCULOSIS 


from  Rabinowitch's  bacillus.  The  points  of  distinction  are  of  a  minor 
character.  Other  more  or  less  similar  bacilli  have  been  cultivated  by 
Tobler,  Coggi,  and  others.1 

Another  bacillus  of  considerable  interest  is  Johne's  bacillus  or  the 
bacillus  of  "chronic  bovine  pseudo-tuberculous  enteritis,"  the  lesions 
produced  by  it  being  corrugated  thickenings  of  the  mucous  membrane, 
especially  of  the  small  intestine.  The  disease  has  now  been  observed  in 
various  countries,  and  several  cases  in  Britain  have  been  recorded  by 
M'Fadyean.  The  bacilli  occur  in  large  numbers  in  the  lesions,  and  can 
readily  be  found  in  scrapings  from  the  surface.  They  resemble  the 

tubercle  bacillus  in  appear- 
ance, but  on  the  whole  are 
rather  shorter ;  they  are 
equally  acid-fast.  The  organ- 
ism has  not  yet  been  culti- 
vated outside  the  body. 

Smegma  Bacillus.— This 
organism  is  of  importance, 
as  in  form  and  staining  re- 
action it  somewhat  resembles 
the  tubercle  bacillus  and  may 
be  mistaken  for  it.  It  occurs 
often  in  large  numbers  in 
the  smegma  prseputiale  and 
in  the  region  of  the  external 
genitals,  especially  where 
there  is  an  accumulation  of 
fatty  matter  from  the  secre- 
tions. Morphologically  it  is 
a  slender  slightly  curved 
organism,  like  the  tubercle 
bacillus  but  usually  dis- 
tinctly shorter  (Fig.  90). 
Like  the  tubercle  bacillus  it 
stains  with  some  difficulty 

and  resists  decolorisation  with  strong  mineral  acids.  Most  observers 
ascribe  the  latter  fact  to  the  fatty  matter  with  which  it  is  surrounded, 
and  find  that  if  the  specimen  is  treated  with  alcohol  the  organism  is 
easily  decolorised.  Czaplewski,  however,  who  claims  to  have  cultivated 
it  on  various  media,  finds  that  in  culture  it  shows  resistance  to  decolor- 
isation both  with  alcohol  and  with  acids,  and  considers,  therefore,  that 
the  reaction  is  not  due  to  the  surrounding  fatty  medium.  We  have 
found  that  in  smegma  it  can  be  readily  decolorised  by  a  minute's  exposure 
to  alcohol  after  the  usual  treatment  with  sulphuric  acid,  and  thus  can 
be  readily  distinguished  from  the  tubercle  bacillus.  We,  moreover, 
believe  that  minor  points  of  difference  in  the  microscopic  appearances  of 
the  two  organisms  are  quite  sufficient  to  make  the  experienced  observer 
suspicious  if  he  .should  meet  with  the  smegma  bacillus  in  urine,  and  lead 
him  to  apply  the  decolorising  test.  Difficulty  will  only  occur  when  a  few 
scattered  bacilli  retaining  the  fuchsin  occur. 

Its   cultivation,    which   is   attended  with  some  difficulty,  was   first 

1  For  further  details  on  this  subject,  vide  Potet,  Etudes  sur  les  bacilles  dites 
acidophiles.  Paris,  1902. 


FIG.  90. — Smegma  bacilli.     Film  preparation 

of  smegma. 
Ziehl-Neelsen  stain.      x  1000. 


ACTION   OF   DEAD   TUBERCLE   BACILLI       255 

effected  by  Czaplewski.  On  serum  it  grows  in  the  form  of  yellowish- 
grey  irregularly  rounded  colonies  about  1  mm.  in  diameter,  sometimes 
becoming  confluent  to  form  a  comparatively  thick  layer.  He  found  that 
it  also  grew  on  glycerin  agar  and  in  bouillon.  It  is  non-pathogenic  to 
various  animals  which  have  been  tested. 

Cowie  has  recently  found  that  acid-fast  bacilli  are  of  common  occur- 
rence in  the  secretions  of  the  external  genitals,  mammae,  etc. ,  in  certain 
of  the  lower  animals,  and  that  these  organisms  vary  in  appearance.  He 
considers  that  the  term  "smegma  bacillus"  probably  represents  a 
number  of  allied  species. 

The  question  may  be  asked — do  these  results  modify  the 
validity  of  the  staining  reaction  of  tubercle  bacilli  as  a  means  of 
diagnosis'?  The  source  of  any  acid-fast  bacilli  in  question  is 
manifestly  of  importance,  and  it  may  be  stated  that  when  these 
have  been  obtained  from  some  source  outside  the  body,  or  where 
contamination  from  without  has  been  possible,  their  recognition 
as  tubercle  bacilli  cannot  be  established  by  microscopic  examina- 
tion alone.  In  the  case  of  material  coming  from  the  interior  of 
the  body,  however, — sputum,  etc., — the  condition  must  be  looked 
on  as  different,  and  although  an  acid-fast  bacillus  (not  tubercle) 
has  been  found  by  Rabinowitch  in  a  case  of  pulmonary  gangrene 
we  have  no  sufficient  data  for  saying  that  acid-fast  bacilli  other 
than  the  tubercle  bacillus  flourish  within  the  tissues  of  the  human 
body  except  in  such  rare  instances  as  to  be  practically  negligible. 
(To  this  statement  the  case  of  the  leprosy  bacillus  is  of  course 
an  exception.)  Accordingly,  up  till  now,  the  microscopic  ex- 
amination of  sputum,  etc.,  cannot  be  'said  to  have  its  validity 
shaken,  and  we  have  the  results  of  enormous  clinical  experience 
that  such  examination  is  practically  of  unvarying  value.  Never- 
theless the  facts  established  with  regard  to  other  acid-fast  bacilli 
must  be  kept  carefully  in  view,  and  great  care  must  be  exercised 
when  only  one  or  two  bacilli  are  found,  especially  if  they  deviate 
in  their  morphological  characters  from  the  tubercle  bacillus. 

Action  of  dead  Tubercle  Bacilli. — The  remarkable  fact  has 
been  established  by  independent  investigators  that  tubercle 
bacilli  in  the  dead  condition,  when  introduced  into  the  tissues 
in  sufficient  numbers,  can  produce  tubercle-like  nodules.  Prudden 
and  Hodenpyl,  by  intravenous  injection  in  rabbits  of  cultures 
sterilised  by  heat,  produced  in  the  lungs  small  nodules  in  which 
giant -cells,  but  no  caseation,  were  occasionally  present,  and 
which  were  characterised  by  more  growth  of  fibrous  tissue  than 
in  ordinary  tubercle.  The  subject  was  very  fully  investigated 
with  confirmatory  results  by  Straus  and  Gamaleia,  who  found 
that,  if  the  number  of  bacilli  introduced  into  the  circulation  were 
large,  there  resulted  very  numerous  tubercle  nodules  with  well- 


256  TUBERCULOSIS 

formed  giant -cells,  and  occasionally  traces  of  caseation.  The 
bacilli  can  be  well  recognised  in  the  nodules  by  the  ordinary 
staining  method.  In  these  experiments  the  bacilli  were  killed 
by  exposure  to  a  temperature  of  115°  C.  for  ten  minutes  before 
being  injected.  Similar  nodules  can  be  produced  by  intra- 
peritoneal  injection.  Subcutaneous  injection,  on  the  other 
hand,  produces  a  local  abscess,  but  in  this  case  no  secondary 
tubercles  are  found  in  the  internal  organs.  Further,  in  many 
of  the  animals  inoculated  by  the  various  methods  a  condition  of 
marasmus  sets  in  and  gradually  leads  to  a  fatal  result,  there 
being  great  emaciation  before  death.  These  experiments,  which 
have  been  confirmed  by  other  observers,  show  that  even  after 
the  bacilli  are  dead  they  preserve  their  staining  reactions  in 
the  tissues  for  a  long  time,  and  also  that  there  are  apparently 
contained  in  the  bodies  of  the  dead  bacilli  certain  substances 
which  act  locally,  producing  proliferative  and,  to  a  less  extent, 
degenerative  changes,  and  which  also  markedly  affect  the  general 
nutrition.  S.  Stockman  has  found  that  an  animal  inoculated 
with  large  numbers  of  dead  tubercle  bacilli  afterwards  gives  the 
tuberculin  reaction. 

Practical  Conclusions. — From  the  facts  above  stated  with 
regard  to  the  conditions  of  growth  of  the  tubercle  bacilli,  their 
powers  of  resistance,  and  the  paths  by  which  they  can  enter  the 
body  and  produce  disease  (as  shown  by  experiment),  the  manner 
by  which  tuberculosis  is  naturally  transmitted  can  be  readily 
understood.  Though  the  experiments  of  Sander  show  that 
tubercle  bacilli  can  multiply  on  vegetable  media  to  a  certain 
extent  at  warm  summer  temperature,  it  is  doubtful  whether  all 
the  conditions  necessary  for  growth  are  provided  to  any  extent 
in  nature.  At  any  rate,  the  great  multiplying  ground  of  tubercle 
bacilli  is  the  animal  body,  and  tubercular  tissues  and  secretions 
containing  the  bacilli  are  the  chief,  if  not  the  only,  means  by 
which  the  disease  is  spread.  The  tubercle  bacilli  leave  the  body 
in  large  numbers  in  the  sputum  of  phthisical  patients,  and  when 
the  sputum  becomes  dried  and  pulverised  they  are  set  free  in 
the  air.  Their  powers  of  resistance  in  this  condition  have  already 
been  stated.  As  examples  of  the  extent  to  which  this  takes 
place,  it  may  be  said  that  their  presence  in  the  air  of  rooms 
containing  phthisical  patients  has  been  repeatedly  demonstrated. 
Williams  placed  glass  plates  covered  with  glycerine  in  the 
ventilating  shaft  of  the  Brompton  Hospital,  and  after  five  days 
found,  by  microscopic  examination,  tubercle  bacilli  on  the  surface, 
whilst  Klein  found  that  guinea-pigs  kept  in  the  ventilating  shaft 
became  tubercular.  Cornet  produced  tuberculosis  in  rabbits  by 


ACTION   OF   DEAD   TUBERCLE   BACILLI       257 

inoculating  them  with  dust  collected  from  the  walls  of  a  con- 
sumptive ward.  Tubercle  bacilli  are  also  discharged  in  consider- 
able quantities  in  the  urine  in  tubercular  disease  of  the  urinary 
tract,  and  also  by  the  bowel  when  there  is  tubercular  ulceration  ; 
but,  so  far  as  the  human  subject  is  concerned,  the  great  means 
of  disseminating  the  bacilli  in  the  outer  world  is  dried  phthisical 
sputum,  and  the  source  of  danger  from  this  means  can  scarcely 
be  over-estimated.  Every  phthisical  patient  ought  to  be  looked 
upon  as  a  fruitful  source  of  infection  to  those  around,  and  should 
only  expectorate  on  to  pieces  of  rag  which  are  afterwards  to  be 
burnt,  or  into  special  receptacles  which  are  to  be  then  sterilised 
either  by  boiling  or  by  the  addition  of  a  5  per  cent  solution  of 
carbolic  acid. 

Another  great  source  of  infection  is  in  all  probability  the 
milk  of  cows  affected  with  tuberculosis  of  the  udder.  In  such 
cases  the  presence  of  tubercle  bacilli  in  the  milk  can  usually  be 
readily  detected  by  centrifugalising  it,  and  then  examining  the 
deposit  microscopically,  or  by  inoculating  an  animal  with  it.  As 
pointed  out  by  Woodhead  and  others,  the  milk  from  cows  thus 
affected  is  probably  the  great  source  of  tabes  mesenterica,  which 
is  so  common  in  young  subjects.  In  these  cases  there  may  be 
tubercular  ulceration  of  the  intestine,  or  it  may  be  absent. 
Woodhead  found  that  out  of  127  cases  of  tuberculosis  in  children, 
the  mesenteric  glands  showed  tubercular  affection  in  100,  and 
that  there  was  ulceration  of  the  intestine  in  43.  It  is  especially 
in  children  that  this  mode  of  infection  occurs,  as  in  the  adult 
ulceration  of  the  intestine  is  rare  as  a  primary  infection,  though 
it  is  common  in  phthisical  patients  as  the  result  of  infection  by 
the  bacilli  in  the  sputum  which  has  been  swallowed.  There  is 
less  risk  of  infection  by  means  of  the  flesh  of  tubercular  animals, 
for,  in  the  first  place,  tuberculosis  of  the  muscles  of  oxen  being 
very  rare,  there  is  little  chance  of  the  bacilli  being  present  in  the 
flesh  unless  the  surface  has  been  smeared  with  the  juice  of  the 
tubercular  organs,  as  in  the  process  of  cutting  up  the  parts ;  and 
in  the  second  place,  even  when  present  they  will  be  destroyed  if 
the  meat  is  thoroughly  cooked. 

We  may  state,  therefore,  that  the  two  great  modes  of  infection 
are  by  inhalation,  and  by  ingestion,  of  tubercle  bacilli.  By  the 
former  method  the  tubercle  bacilli  will  in  most  cases  be  derived 
from  the  human  subject ;  in  the  latter,  probably  from  tubercular 
cows,  though  inhaled  tubercle  bacilli  may  also  be  swallowed  and 
contamination  of  food  by  tubercular  material  from  the  human 
subject  may  occur.  Alike  when  inhaled  and  when  ingested, 
tubercle  bacilli  may  lodge  about  the  pharynx  and  thus  come  to 
17 


258  TUBERCULOSIS 

infect  the  pharyngeal  lymphoid  tissue,  tonsils,  etc.,  tubercular 
lesions  of  these  parts  being  much  more  frequent  than  was 
formerly  supposed.  Thence  the  cervical  lymphatic  glands  may 
become  infected,  and  afterwards  other  groups  of  glands,  bones, 
or  joints,  and  internal  organs. 

The  Toxins  of  the  Tubercle  Bacillus. — Two  outstanding 
features  of  the  action  of  the  tubercle  bacillus  are  the  occurrence 
of  necrosis  in  the  cells  of  tubercle  nodules  and  the  production 
of  general  disturbances  of  metabolism  accompanied  by  fever. 
It  is  natural  to  refer  these  phenomena  to  the  effects  of  toxins 
formed  by  the  organism.  The  study  of  such  toxins  centres 
round  the  substance  known  as  tuberculin  which  Koch  brought 
forward  in  1890-1  as  a  curative  agent  for  tubercular  affections. 

Koch's  Tuberculin. — Koch  stated  that  if  in  a  guinea-pig 
suffering  from  the  effects  of  a  subcutaneous  inoculation  with 
tubercle  bacilli,  a  second  subcutaneous  inoculation  of  tubercle 
bacilli  was  practised  in  another  part  of  the  body,  superficial 
ulceration  occurred  in  the  primary  tubercular  nodule,  the  wound 
healed,  and  the  animal  did  not  succumb  to  tuberculosis.  This 
reaction  was  further  studied  by  means  of  tuberculin,  which 
consisted  of  a  concentrated  glycerin  bouillon  culture  of  tubercle 
in  which  the  bacilli  had  been  killed  by  heat.  Its  essential 
components  probably  were  the  dead  and  often  macerated  bacilli 
and  the  substances  indestructible  by  boiling  which  existed  in 
these  bacilli,  or  which  were  formed  during  their  growth.  The 
injection  of  '25  c.c.  of  tuberculin  into  a  healthy  man  causes,  in 
from  three  to  four  hours,  malaise,  tendency  to  cough,  laboured 
breathing,  and  moderate  pyrexia ;  all  of  which  pass  off  in 
twenty-four  hours.  The  injection  (the  site  of  the  injection  being 
quite  unimportant),  however,  of  '01  c.c.  into  a  tubercular  person 
gives  rise  to  similar  symptoms,  but  in  a  much  more  aggravated 
form,  and  in  addition  there  occurs  around  any  tubercular  focus 
great  inflammatory  reaction,  resulting  in  necrosis  and  a  casting 
off  of  the  tubercular  mass,  when  this  is  possible,  as  for  instance 
in  the  case  of  lupus.  The  bacilli  are,  it  was  shown,  not  killed  in 
the  process. 

Koch's  theory  of  the  action  of  the  substance  was  that  the  tubercle 
bacillus  ordinarily  secretes  a  body  having  a  necrotic  action  on  the  tissues. 
When  this  is  injected  into  a  tubercular  patient,  the  proportion  present 
round  a  tubercular  focus  is  suddenly  increased,  inflammatory  reaction 
takes  place  around,  and  necrosis  of  the  spreading  margin  occurs  very 
rapidly,  the  material  containing  the  living  or  dead  bacilli  being  thrown 
off  en  masse  instead  of  being  disintegrated  piecemeal.  It  appears, 
however,  that  this  explanation  may  not  be  the  true  one  ;  for,  on  the  one 
hand,  other  substances  besides  products  of  the  tubercle  bacillus  may 


TOXINS   OF   THE   TUBERCLE   BACILLUS      259 

give  rise  to  similar  effects  in  tubercular  animals,  and,  on  the  other,  a 
similar  reaction  can  take  place  in  other  diseases  where  there  is  locally  in 
the  body  a  deposit  of  new  tissue.  Matthes  has,  for  instance,  found  that 
albtimoses  and  peptones  isolated  from  the  ordinary  peptic  digestion  of 
various  albumins  give  the  same  reaction  in  tubercular  guinea-pigs.  The 
injection  of  milk,  lactic  acid,  ricin,  all  give  a  similar  result.  Before  the 
discovery  of  tuberculin,  Gamaleia  had  found  that  tubercular  animals 
were  very  susceptible  to  the  toxins  of  the  vibrio  Metchnikovi ;  and  later 
Metchnikoff  found  that  a  similar  susceptibility  existed  towards  the 
toxins  of  the  bacillus  of  fowl  cholera.  How  complicated  the  tuberculin 
reaction  is  is  shown  by  the  fact  that  a  similar  reaction  has  taken  place 
when  tuberculin  has  been  injected  into  persons  suffering  from  diseases 
other  than  tubercle,  e.g.  cancer,  sarcoma,  syphilis. 

The  hopes  which  the  introduction  of  tuberculin  raised,  that  a 
curative  agent  against  tuberculosis  had  been  discovered,  were  soon 
found  not  to  be  justified.  It  was  very  difficult  to  see  how  the 
necrosed  material  which  was  produced  and  which  contained  the 
still  living  bacilli,  could  be  got  rid  of  either  naturally,  as  would 
be  necessary  in  the  case  of  a  small  tubercular  deposit  in  a  lung 
or  a  lymphatic  gland,  or  artificially,  as  in  a  complicated  joint- 
cavity  where  surgical  interference  could  be  undertaken.  Not 
only  so,  but  the  ulceration  which  might  be  the  sequel  of  the 
necrosis  appeared  to  open  a  path  for  fresh  infection.  Soon  facts 
were  reported  which  justified  these  criticisms.  Cases  where 
rapid  acute  tubercular  conditions  ensued  on  the  use  of  tuberculin 
were  reported,  arid  in  a  few  months  the  treatment  was  practically 
abandoned. 

The  Use  of  Tuberculin  in  the  Diagnosis  of  Tuberculosis  in  Cattle. — 
This  is  now  the  chief  use  to  which  tuberculin  is  put.  In  cattle, 
tuberculosis  may  be  present  without  giving  rise  to  apparent 
symptoms.  It  is  thus  important  from  the  point  of  view  of  human 
infection  that  an  early  diagnosis  should  be  made.  The  method  is 
applied  as  follows  : — The  animals  are  kept  twenty-four  hours  in  their 
stalls,  and  the  temperature  is  taken  every  three  hours,  from  four  hours 
before  the  injection  till  twenty-four  after.  The  average  tempera- 
ture in  cattle  is  102 '2°  F.  ;  30  to  40  centigrammes  of  tuberculin  are 
injected,  and  if  the  animal  be  tubercular  the  temperature  rises  2°  or  3°  F. 
in  eight  to  twelve  hours  and  continues  elevated  for  ten  to  twelve  hours. 
Bang,  who  has  worked  most  at  the  subject,  lays  down  the  principle  that 
the  more  nearly  the  temperature  approaches  104°  F.  the  more  reason  for 
suspicion  is  there.  He  gives  a  record  of  280  cases  where  the  value  of 
the  method  was  tested  by  subsequent  post-mortem  examination.  He 
found  that  with  proper  precautions  the  error  was  only  3 '3  per  cent. 
The  method  has  been  largely  practised  in  all  parts  of  the  world,  and  is  of 
great  value. 

While  it  is  undoubted  that  tuberculin  contains  toxic  products 
formed  by  the  bacilli,  we  know  nothing  of  the  nature  of  the 
toxins  present.  From  the  fact  that  filtered  cultures  cause  little 


260  TUBERCULOSIS 

toxic  effect,  and  that  trituration  of  the  bacilli  increases  the 
poisonous  content  of  a  culture,  it  is  inferred  that  we  have  to  deal 
with  endotoxins,  but  beyond  this  statement  we  cannot  go. 
Hitherto  no  success  has  attended  attempts  to  gain  a  closer 
knowledge  of  the  nature  of  such  substances.  It  has  been  stated 
that  albumoses  of  a  special  kind  are  present  in  tuberculin,  but 
nothing  definite  has  emerged  from  the  investigation  of  these 
bodies. 

Active  Immunisation  against  the  Tubercle  Bacillus.— 
Koch's  Tuberculin-R.  Our  knowledge  here  centres  round  the 
substance  introduced  by  Koch  in  1897  under  the  name  of 
"Tuberculin-R,"  or  the  new  tuberculin.  Koch's  new  researches 
consisted  (1)  of  an  attempt  to  immunise  animals  against  the 
tubercle  bacillus  by  employing  its  intracellular  toxins  ;  (2)  of 
trying  to  utilise  such  an  immunisation  to  aid  the  tissues  of  an 
r.nimal  already  attacked  with  tubercle  the  better  to  combat  the 
effects  of  the  bacilli.  The  method  of  obtaining  the  intracellular 
toxins  was  as  follows.  Bacilli  from  young  virulent  cultures  were 
dried  in  vacuo,  and  disintegrated  in  an  agate  mill,  treated  with 
distilled  water  and  centrifugalised.  The  clear  fluid  was  decanted, 
and  is  called  by  Koch  "  Tuberculin-O."  The  remaining  deposit 
was  again  dried,  ground,  treated  with  water  and  centrifugalised, 
the  clear  fluid  being  again  decanted,  and  this  process  was 
repeated  with  successive  residues  till  no  residue  remained. 
These  fluids  put  together  constitute  the  "  Tuberculin-R." 

From  the  fact  that  tuberculin-O  gave  no  cloudiness  when 
glycerin  was  added,  Koch  concluded  that  it  contained  the  sub- 
stances present  in  the  glycerin-bouillon  extracts  originally  used 
by  him,  and  he  held  this  was  borne  out  by  the  readiness  with 
which  a  tuberculin  reaction  could  be  caused  by  it.  Similarly,  as 
tuberculin-R  gave  a  cloudiness  with  glycerin  and  did  not  readily 
originate  a  reaction,  he  considered  that  it  contained  different 
products  of  the  bacillus.  When  injected  into  animals  in 
repeated  and  increasing  doses,  T^  mgrm.  being  the  initial  dose, 
tuberculin-R  is  said  to  produce  immunity  against  the  original 
extract,  against  tuberculin-O,  and  against  living  and  virulent 
tubercle  bacilli.  Another  preparation  has  also  been  introduced 
known  as  "  Koch's  new  tuberculin  "  (Bazillenemulsion).  This  is 
an  emulsion  of  ground  tubercle  bacilli  in  water  containing  50  per 
cent  of  glycerin ;  it  thus  really  contains  both  tuberculin-O  and 
tuberculin-R.  Both,  especially  tuberculin-R,  have  been  used  for 
the  treatment  of  tuberculosis  in  man,  especially  for  early  localised 
lesions.  In  the  case  of  both  substances  commencing  with  from 
to  5-^jQ-  mgrm,  gradually  increasing  doses  were  given  every 


OPSONINS   IN   TUBERCULOSIS  261 

second  day,  and  the  rule  originally  laid  down  for  the  regulation 
of  the  dosage  was  that  no  amount  should  be  given  which  raised 
the  temperature  more  than  *5°  F.  Very  various  opinions  have 
been  expressed  as  to  the  efficacy  of  such  treatment.  There  is 
little  doubt  that  in  certain  cases  of  local  conditions,  such  as  lupus, 
tubercular  joints,  glands  and  genito-urinary  tuberculosis,  improve- 
ment has  followed  its  application ;  but  where  febrile  conditions 
indicate  that  general  disturbances  are  in  existence,  there  is  little 
or  no  justification  for  its  being  applied,  and  even  in  many  local 
conditions  the  absence  of  benefit  is  so  marked  that  by  many 
physicians  the  method  has  been  abandoned. 

Active  Immunisation  associated  with  Opsonic  Observations. — • 
Within  recent  years  attention  has  been  directed  to  the  possibility 
of  controlling  the  use  of  tuberculin-R  by  observations  of  its 
effect  on  the  opsonic  qualities  of  the  serum.  Wright,  early  in 
his  work,  showed  that  tubercle  bacilli  when  sensitised  by  an 
appropriate  serum,  were  readily  phagocyted  by  the  polymorpho- 
nucleate  leucocytes,  and  the  relative  sensitising  capacities  of 
serum  from  tubercular  and  non-tubercular  cases  has  been  widely 
studied.  According  to  Wright,  in  strictly  localised  tuberculosis 
the  opsonic  index  is  persistently  low,  varying  from  '1  to  '9, 
while  in  tubercle  with  general  disturbances  it  fluctuates  greatly 
from  day  to  day,  being  sometimes  below,  sometimes  above  unity. 
To  take  the  former  and  simpler  case,  he  holds  that  if  the  treat- 
ment with  injections  of  tuberculin-R  be  controlled  by  noting  the 
effect  produced  on  the  opsonic  index,  great  improvement  in  the 
patient's  condition  may  result.  Wright's  interpretation  of  what 
occurs  is  bound  up  with  his  views  on  the  nature  of  the  effects 
produced.  These  views  are  briefly  as  follows.  For  'reasons 
unknown  the  opsonic  qualities  of  the  body  fluids  may  become 
abnormally  low,  and  the  tubercle  bacilli,  if  they  gain  admission  to 
the  body,  can  multiply  locally.  This  multiplication  is  associated 
with  a  still  further  local  diminution  of  the  opsonins.  By  the 
introduction  of  such  a  substance  as  Koch's  tuberculin-R  the 
bodily  mechanism,  whatever  it  is,  which  produces  the  opsonins 
is  stimulated,  and  a  rise  in  the  general  opsonic  index  occurs. 
Naturally  this  is  accompanied  by  a  passing  to  the  site  of  infec- 
tion of  fluids  more  rich  in  opsonins  than  previously,  the  activity 
of  the  phagocytes  comes  into  play  and  the  tubercle  bacilli  are 
destroyed.  But  any  such  vaccination  process  must  be  controlled 
by  constant  observations  of  the  opsonic  index,  and  it  is  only  by 
this  means  that  not  only  good  results  can  be  obtained,  but  that 
the  production  of  harmful  effects  can  be  prevented.  The  reason 
of  this  is  that  in  a  great  many  cases  the  injection  of  a  bacterial 


262  TUBEECULOSIS 

vaccine  is  followed  by  a  decrease  in  the  opsonic  qualities  of  the 
serum, — the  occurrence  of  a  negative  phase.  During  such  a 
period  of  depression  there  is  probably  an  increased  susceptibility 
to  the  action  of  the  bacilli.  Now,  in  order  to  get  permanent 
benefit  from  the  vaccination  process,  repeated  injections  of  the 
tuberculin  must  be  practised,  and  if  an  injection  be  given  during 
a  negative  phase,  actual  harm  may  be  done.  The  course  of  a 
successful  vaccination  is  that,  after  the  passing  off  of  the  negative 
phase,  the  opsonic  index  should  rise  to  above  its  original  level, 
— the  occurrence  of  a  positive  phase.  It  is  when  this  positive 
phase  is  fully  developed  that  a  fresh  inoculation  can  be  practised 
with  success.  The  new  negative  phase  which  will  now  occur 
may  not  cause  a  drop  to  below  the  level  of  the  original  state  of 
the  serum,  and  the  hope  is  that  its  succeeding  positive  phase 
will  carry  the  opsonic  index  still  higher  and  ensure  a  still  greater 
resistance  to  the  bacterium.  The  importance  of  the  observations 
of  the  opsonic  index  lies  in  this  that  in  antibacterial  vaccina- 
tions the  degree  of  active  immunisation  which  can  be  attained  is 
always  much  less  than  is  the  case  with  immunisation  against 
such  a  substance  as  the  diphtheria  toxin,  although  in  the  latter 
there  also  occur  negative  and  positive  phases  of  a  precisely 
similar  character.  If  an  injection  be  practised  during  a  negative 
phase,  then  a  still  further  drop  in  the  opsonic  content  of  the 
serum  will  occur  and  a  fresh  growth  of  the  invading  bacilli  is 
likely.  There  are  very  great  variations  in  the  capacities  shown 
by  tubercular  patients  to  react  to  a  vaccination  process.  In  certain 
cases  good  positive  phases  are  readily  and  quickly  produced,  while 
in  others  after  an  inoculation  the  negative  phase  is  long  con- 
tinued and  may  even  show  no  tendency  to  pass  into  a  positive 
phase.  The  irregularities  in  the  opsonic  index  in  cases  where 
there  is  a  general  disturbance  of  metabolism  Wright  explains  by 
supposing  that  they  result  from  very  irregular  auto-infections  of 
the  patient's  body  by  tubercular  products  from  the  local  lesions, 
— positive  and  negative  phases  being  produced  without  the  pur- 
posive quality  which  ought  to  characterise  a  successful  therapeutic 
vaccination.  Such  auto-infections  may  come  about  in  various 
ways,  and  Wright  is  of  opinion  that  exercise,  for  instance,  may 
disseminate  both  tubercular  products  and  tubercle  bacilli, — he 
having  noticed  in  tubercle  patients  a  fall  in  the  opsonic  index 
after  muscular  exertion. 

With  regard  to  the  details  of  the  immunisation,  Wright's 
chief  point  is  that  the  repeated,  uncontrolled  injections  of  tuber- 
culin such  as  were  originally  given  may  very  likely  have  a  harmful 
result,  and  that  when  an  injection  is  practised  it  is  not  necessary 


OPSONINS   IN   TUBERCULOSIS  263 

for  constitutional  effects  to  occur  in  order  that  a  beneficial  result 
may  follow.  Hence  much  smaller  doses  of  tuberculin  than 
hitherto  are  given  by  him.  For  ordinary  cases  with  low  opsonic 
index  and  no  evidence  of  constitutional  disturbance,  an  amount 
of  tuberculin  corresponding  to  from  one-thousandth  to  a  six- 
hundredth  of  a  millegramme  of  tubercle  powder  is  a  sufficient 
dose,  and  if  any  dose  seems  to  produce  a  pronounced  negative 
phase  then  a  smaller  dose  ought  to  be  tried  at  the  next  inocula- 
tion. For  cases  clinically  tubercular  where  the  index  is  about 
normal,  then  smaller  doses,  say,  the  equivalent  of  a  two- 
thousandth  of  a  millegramme  or  less  ought  to  be  used, — the 
effect  on  the  index  being  carefully  watched.  In  any  case,  the 
dose  which  is  found  to  give  the  highest  positive  phase  is  the 
optimum  dose  and  one  which  need  not  necessarily  be  increased. 
Cases  where  there  is  constitutional  disturbance  should  be  as  a 
rule  left  untreated. 

The  general  position  of  Wright  and  his  school  is,  that  it  is 
only  by  the  observation  of  the  opsonic  index  that  the  application 
of  the  tuberculin  treatment  can  be  effectually  controlled, — 
deductions  based  on  clinical  data,  such  as  absence  of  interference 
with  pulse  rate,  temperature,  etc.,  or  increase  of  body  weight 
after  an  inoculation  being  unreliable,  and^further  evidence  of  the 
unreliability  of  such  tests  is  brought  forward  in  the  fact  that,  in 
cases  of  apparent  benefit  from  sanatorium  treatment,  the  opsonic 
index  may  still  be  very  low.  With  regard  to  the  results 
obtained,  many  cases  have  been  brought  forward  by  Wright  and 
others  where  benefit  has  followed  the  putting  into  practice  of  the 
principles  enunciated,  and  there  is  little  doubt  that  the  work 
done  has  given  a  fresh  start  to  the  active  immunisation  method 
in  the  treatment  of  tuberculosis.  An  outstanding  event  of 
Wright's  work  in  this  field  has  been  his  insistance  on  the  good 
effects  produced  by  extremely  small  doses  of  tuberculin  (down  to 
the  four-thousandth  of  a  millegramme)  given  at  fairly  long  inter- 
vals (say  10  days  or  more).  With  regard  to  the  efficacy  of  the 
opsonic  method  as  affording  an  index  to  the  progress  of  a  case 
it  must  be  recognised  that  the  method  is  still  on  its  trial,  and 
it  is  doubtful  if  even  in  the  work  of  the  most  careful  observers 
the  limits  of  the  experimental  error  of  the  opsonic  method  have 
been  sufficiently  defined. 

The  whole  question  of  immunisation  against  the  tubercle 
bacillus  presents  many  difficulties,  and  it  is  the  merit  of  Wright's 
work  that  it  has  shed  fresh  light  on  some  of  these.  One  great 
difficulty  arises  from  the  great  chronicity  of  the  results  of  the 
infection  in  the  majority  of  human  cases.  It  is  probably 


264  TUBERCULOSIS 

true  not  only  of  man  but  of  many  species  of  animals  used  in 
experimental  inquiries,  that  many  individuals  are  on  the  border- 
line between  immunity  and  susceptibility.  From  the  wide 
spread  of  the  bacilli  in  centres  of  human  population,  it  is  certain 
that  the  opportunity  for  infection  arises  in  a  very  large  propor- 
tion of  the  race  ;  in  many  cases  no  results  follow  infection,  and 
in  many  others  small  lesions  occur  which  do  not  develop  further ; 
this  has  actually  been  shown  by  morbid  anatomists  to  be  the 
case.  The  disease  being  thus  so  often  characterised  by  transient 
local  effects  without  constitutional  disturbance,  the  course  of  an 
immunisation  may  be  expected  to  be  rather  different  from  that 
obtaining  in  an  ordinary  acute  affection,  though  the  underlying 
processes  may  be  of  the  same  nature.  It  is  difficult,  for  instance, 
on  account  of  the  slowness  of  tubercular  processes,  to  define 
recovery  from  an  attack  of  the  disease,  or  to  speak  of  an  animal 
recovering  from  the  effect  of  an  inoculation  during  an  immunisa- 
tion. It  follows  that  little  is  known  regarding  an  attenuation 
of  the  tubercle  bacillus  analogous  to  what  is  an  important 
feature  in  immunisations  against  other  organisms.  It  has 
been  thought  by  some  that  the  tubercle  bacilli  from  so-called 
scrofulous  glands  are  less  virulent  than  those,  say,  from  phthisis, 
but  apparently  here  sufficient  attention  has  not  been  paid  to  the 
difference  of  the  numbers  of  bacilli  injected  in  each  case,  and 
this  appears  to  be  a  very  important  point.  Experiments  have 
also  been  brought  forward  which  appear  to  show  that  the  injec- 
tion of  bacilli  from  avian  tuberculosis  could  protect  the  dog 
against  bacilli  derived  from  man.  But  these  are  not  yet  conclusive. 

Agglutinative  Phenomena. — The  serum  of  tubercular  patients 
has  been  found  to  exert  an  agglutinating  action  on  the  tubercle 
bacillus.  A  convenient  method  is  to  add  different  amounts 
of  serum,  commencing  with,  say,  *1  c.c.,  to  quantities  of  a 
dilution  of  the  new  tuberculin  (Bazillenemulsion)  equivalent  to 
1  part  of  the  bacterial  bodies  to  10,000  of  diluent,  and  leave  the 
mixture  for  24  hours  before  observing.  As  with  other  agglutina- 
tive observations,  it  is  difficult  to  correlate  the  degree  of  agglu- 
tinating power  of  the  serum  with  the  degree  of  immunisation 
possessed  by  the  individual  from  which  it  was  derived. 

Anti tubercular  Sera. — Several  attempts  have  been  made  to 
treat  tuberculosis  with  the  serum  of  animals  immunised  by  the 
tubercle  bacillus  or  its  products.  The  most  successful  is  perhaps 
that  of  Maragliano.  This  author  distinguishes  between  the 
toxic  materials  contained  in  the  bodies  of  the  bacilli  (which 
withstand,  unchanged,  a  temperature  of  100°  C.)  and  those 
secreted  into  the  culture  fluid  (which  are  destroyed  by  heat). 


METHODS   OF   EXAMINATION  265 

The  substance  used  by  him  for  immunising  his  animals  consists 
of  three  parts  of  the  former  and  one  of  the  latter.  The  animals 
employed  are  the  dog,  the  ass,  the  horse.  The  serum  obtained 
from  these  is  capable  of  protecting  healthy  animals  against  an 
otherwise  fatal  dose  of  tuberculin,  but  very  little  importance  can 
be  attached  to  this  result.  Maragliano  does  not  appear  to  have 
studied  the  effects  of  this  serum  on  tubercular  animals,  but  it 
has  been  tried  in  a  great  number  of  cases  of  human  tuberculosis, 
2  c.c.  being  injected  subcutaneously  every  two  days.  Improve- 
ment is  said  to  have  taken  place  in  a  certain  proportion,  especi- 
ally of  mild  non-febrile  cases. 

An  antitubercular  serum  has  also  been  introduced  by 
Marmorek.  This  observer  considers '  that  the  tubercle  bacillus 
cannot  produce  in  ordinary  media  the  toxins  which  it  originates 
when  exposed  to  the  antagonism  of  the  bodily  cells.  He  tries 
to  make  good  this  defect  by  first  growing  it  in  a  serum  antago- 
nistic to  some  of  the  phagocytic  cells  of  the  body ;  for  this  a 
leucotoxic  serum  is  used.  When  the  bacillus  has  grown  in  this 
presumably  favourable  soil  it  is  transferred  to  a  medium  con- 
taining a  substance  which  may  be  unfavourable  :  and  for  this 
there  is  employed  a  medium  containing  liver  extract,  the 
liver  being  an  organ  in  which  in  man  tubercular  lesions  are 
comparatively  rare.  The  bacilli  being  thus  accustomed  to  an 
unfavourable  surrounding  are  used  for  immunising  animals,  the 
serum  of  which  is  now  suitable  for  the  treatment  of  human  tuber- 
culosis. It  is  too  soon  to  speak  of  the  effects  of  this  line  of 
treatment. 

Methods  of  Examination. — (1)  Microscopic  Examination. 
Tuberculosis  is  one  of  the  comparatively  fe"w  diseases  in  which  a 
diagnosis  can  usually  be  definitely  made  by  microscopic  examina- 
tion alone.  In  the  case  of  sputum,  one  of  the  yellowish 
fragments  which  are  often  present  ought  to  be  selected ;  dried 
films  are  then  prepared  in  the  usual  way  and  stained  by  the 
Ziehl-Neelsen  method  (p.  101).  In  the  case  of  urine  or  other 
fluids  a  deposit  should  first  be  obtained  by  centrifugalising  a 
quantity  in  a  test-tube,  or  by  allowing  the  fluids  to  stand  in  a 
tall  glass  vessel  (an  ordinary  burette  is  very  convenient).  Film 
preparations  are  then  made  with  the  deposit  and  treated  as 
before.  If  a  negative  result  is  obtained  in  a  suspected  case, 
repeated  examination  should  be  undertaken.  To  avoid  risk  of 
contamination  with  the  smegma  bacillus  the  meatus  of  the 
urethra  should  be  cleansed  and  the  urine  first  passed  should  be 
rejected,  or  the  urine  may  be  drawn  off  with  a  sterile  catheter. 
As  stated  above  it  is  only  exceptionally  that  difficulty  will  arise 


266  TUBERCULOSIS 

to  the  experienced  observer  from  this  cause.     (For  points  to  be 
attended  to,  vide  p.  255.) 

(2)  Inoculation.  —  The    guinea-pig    is    the     most    suitable 
animal.     If  the  material  to  be  tested  is  a  fluid    it  is  injected 
subcutaneously  or  into  the  peritoneum ;  if  solid  or  semi-solid  it 
is  placed  in  a  small  pocket  in  the  skin,  or  it  may  be  thoroughly 
broken  up    in  sterile  water   or  other  fluid   and  the   emulsion 
injected.     By  this  method,  material  in  which  no  tubercle  bacilli 
can  be  found  microscopically  may  sometimes  be  shown  to  be 
tubercular. 

(3)  Cultivation. — Owing  to    the  difficulties  this  is  usually 
quite  impracticable  as  a  means  of  diagnosis,  and  it  is  also  un- 
necessary.    The   best   method    to    obtain    pure   cultures   is   to 
produce  tuberculosis  in  a  guinea-pig  by  inoculation  with  tubercular 
material,  and  then,  killing  the  animal  after  four  or  five  weeks, 
to  inoculate  tubes  of  solidified  blood  serum,  under  strict  aseptic 
precautions,  with  portions  of  a  tubercular  organ,  e.g.  the  spleen. 
The  portions  of  tissue  should  be  fairly  large,  and  should  be  well 
rubbed  into  the  broken  surface  of  the  medium. 


CHAPTER  X. 

LEPROSY. 

LEPROSY  is  a  disease  of  great  interest,  alike  in  its  clinical  and 
pathological  aspects ;  whilst  from  the  bacteriological  point  of 
view  also,  it  presents  some  striking  peculiarities.  The  invariable 
association  of  large  numbers  of  characteristic  bacilli  with  all 
leprous  lesions  is  a  well-established  fact,  and  yet,  so  far,  attempts 
to  cultivate  the  bacilli  outside  the  body,  or  to  produce  the  disease 
experimentally  in  animals,  have  been  attended  with  failure. 
Leprosy,  so  far  as  is  known,  is  a  disease  which  is  confined  to  the 
human  subject,  but  it  has  a  very  wide  geographical  distribution. 
It  occurs  in  certain  parts  of  Europe — Norway,  Russia,  Greece, 
etc.,  but  is  commonest  in  Asia,  occurring  in  Syria,  Persia,  etc. 
It  is  prevalent  in  Africa,  being  especially  found  along  the  coast, 
in  the  Pacific  Islands,  in  the  warmer  parts  of  North  and  South 
America,  and  also  to  a  small  extent  in  the  northern  part  of  North 
America.  In  all  these  various  regions  the  disease  presents  the 
same  general  features,  and  the  study  of  its  pathological  and 
bacteriological  characters,  wherever  such  has  been  carried  on,  has 
yielded  similar  results. 

Pathological  Changes. — Leprosy  is  characteristically  a  chronic 
disease,  in  which  there  is  a  great  amount  of  tissue  change,  with 
comparatively  little  necessary  impairment  of  the  general  health. 
In  other  words,  the  local  effects  of  the  bacilli  are  well  marked, 
often  extreme,  whilst  the  toxic  phenomena  are  proportionately  at 
a  minimum. 

There  are  two  chief  forms  of  leprosy.  The  one,  usually  called 
the  tubercular  form — lepra  tuberosa  or  tuberculosa — is  character- 
ised by  the  growth  of  granulation  tissue  in  a  nodular  form  or  as  a 
diffuse  infiltration  in  the  skin,  in  mucous  membranes,  etc.,  great 
disfigurement  often  resulting.  In  the  other  form,  the  anaesthetic, 
— maculo- anaesthetic  of  Hansen  and  Looft — the  outstanding 

267 


268 


LEPROSY 


changes  are  in  the  nerves,  with  consequent  anaesthesia,  paralysis 
of  muscles,  and  trophic  disturbances. 

In  the  tubercular  form  the  disease  usually  starts  with  the 
appearance  of  erythematous  patches  attended  by  a  small  amount 
of  fever,  and  these  are  followed  by  the  development  of  small 
nodular  thickenings  in  the  skin,  especially  of  the  face,  of  the 
backs  of  hands  and  feet,  and  of  the  extensor  aspects  of  arms  and 


FIG.  91. — Sections  through  leprous  skin,  showing  the  masses  of  cellular 
granulation  tissue  in  the  cutis  ;  the  dark  points  are  clumps  of  bacilli  deeply 
stained. 

Paraffin  section  ;  Ziehl-Neelsen  stain,      x  80. 

legs.  These  nodules  enlarge  and  produce  great  distortion  of  the 
surface,  so  that,  in  the  case  of  the  face,  an  appearance  is  produced 
which  has  been  described  as  "leonine."  The  thickenings  occur 
chiefly  in  the  cutis  (Fig.  91),  to  a  less  extent  in  the  subcutaneous 
tissue.  The  epithelium  often  becomes  stretched  over  them, 
and  an  oozing  surface  becomes  developed,  or  actual  ulceration 
may  occur.  The  cornea  and  other  parts  of  the  eye,  the  mucous 
membrane  of  the  mouth,  larynx,  and  pharynx,  may  be  the  seat 
of  similar  nodular  growths.  Internal  organs,  especially  the 
spleen,  liver,  and  testicles,  may  become  secondarily  affected.  In 


BACILLUS   OF   LEPROSY  269 

all  situations  the  change  is  of  the  same  nature, — a  chronic 
inflammatory  condition  attended  by  abundant  formation  of 
granulation  tissue,  nodular  or  diffuse  in  its  arrangement.  In  this 
tissue  a  large  proportion  of  the  cells  are  of  rounded  or  oval  shape, 
like  hyaline  leucocytes ;  a  number  of  these  may  be  of  compara- 
tively large  size,  and  may  show  vacuolation  of  their  protoplasm 
and  a  vesicular  type  of  nucleus.  These  are  often  known  as 
"  lepra-cells."  Amongst  the  cellular  elements  there  is  a  varying 
amount  of  stroma,  which  in  the  earlier  lesions  is  scanty  and 
delicate,  but  in  the  older  lesions  may  be  very  dense.  Periarteritis 
is  a  common  change,  and  very  frequently  the  superficial  nerves 
become  involved  in  the  nodules  and  undergo  atrophy.  The 
tissue  in  the  leprous  lesions  is  comparatively  vascular,  at  least 
when  young,  and,  unlike  tubercular  lesions,  never  shows  caseation. 
Some  of  the  lepra  cells  may  contain  several  nuclei,  but  we  do 
not  meet  with  cells  resembling  in  their  appearance  tubercle 
giant-cells,  nor  does  an  arrangement  like  that  in  tubercle  follicles 
occur. 

In  the  anaesthetic  form  the  lesion  of  the  nerves  is  the  out- 
standing feature.  These  are  the  seat  of  diffuse  infiltrations 
which  lead  to  the  destruction  of  the  nerve  fibres.  In  the  earlier 
stages,  in  which  the  chief  symptoms  are  pains  along  the  nerves, 
there  occur  patches  on  the  skin,  often  of  considerable  size,  the 
margins  of  which  show  a  somewhat  livid  congestion.  Later, 
these  patches  become  pale  in  the  central  parts,  and  the  periphery 
becomes  pigmented.  There  then  follow  remarkable  series  of 
trophic  disturbances  in  which  the  skin,  muscles,  and  bones  are 
especially  involved.  The  skin  often  becomes  atrophied,  parch- 
ment-like, and  anaesthetic  ;  frequently  pemphigoid  bullse  or  other 
skin  eruptions  occur.  Partly  owing  to  injury  to  which  the  feet 
and  arms  are  liable  from  their  anaesthetic  condition,  and  partly 
owing  to  trophic  disturbances,  necrosis  and  separation  of  parts 
are  liable  to  occur.  In  this  way  great  distortion  results.  The 
lesions  in  the  nerves  are  of  the  same  nature  as  those  described 
above,  that  is,  they  are  the  result  of  a  chronic  inflammatory 
process,  but  the  granulation  tissue  is  scantier,  and  has  a  greater 
tendency  to  undergo  cicatricial  contraction.  This  is  to  be 
associated  with  the  fact  that  the  bacilli  are  present  in  fewer 
numbers. 

Bacillus  of  Leprosy. — This  bacillus  was  first  observed  in 
leprous  tissues  by  Hansen  in  1871,  and  was  the  subject  of  several 
communications  by  him  in  1874  and  later.  Further  researches, 
first  by  Neisser  in  1879,  and  afterwards  by  observers  in 
various  parts  of  the  world,  agreed  in  their  main  results,  and 


270  LEPROSY 

confirmed  the  accuracy  of  Hansen's  observations.  The  bacilli  as 
seen  in  scrapings  of  ulcerated  leprous  nodules,  or  in  sections, 
have  the  following  characters.  They  are  thin  rods  of  practically 
the  same  size  as  tubercle  bacilli,  which  they  also  resemble  both 
in  appearance  and  in  staining  reaction.  They  are  straight  or 
slightly  curved,  and  usually  occur  singly,  or  two  may  be  attached 
end  to  end ;  but  they  do  not  form  chains.  When  stained  they 

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FIG.  92. — Superficial  part  of  leprous  skin  ;  the  cells  of  the  granulation  tissue 
appear  as  dark  patches,  owing  to  the  deeply-stained  bacilli  in  their  interior. 
In  the  upper  part  a  process  of  epithelium  is  seen. 

Paraffin  section  ;  stained  with  carbol-fuchsin  and  Bismarck-brown,     x  500. 

may  have  a  uniform  appearance,  or  the  protoplasm  may  be 
fragmented,  so  that  they  appear  like  short  rows  of  cocci.  They 
often  appear  tapered  at  one  or  both  extremities ;  occasionally 
there  is  slight  club-like  swelling.  Degenerated  and  partially 
broken  down  forms  are  also  seen.  They  take  up  the  basic 
aniline  stains  more  readily  than  tubercle  bacilli,  but  in  order 
to  stain  them  deeply  a  powerful  stain,  such  as  carbol-fuchsin, 
is  necessary.  When  stained,  they  strongly  resist  decolorising, 
though  they  are  more  easily  decolorised  than  tubercle  bacilli. 
The  best  method  is  to  stain  with  carbol-fuchsin  as  for  tubercle 


POSITION   OF   THE   BACILLI  271 

bacilli,  but  to  use  a  weaker  solution  of  sulphuric  acid,  say  5  per 
cent,  in  decolorising  ;  in  the  case  of  films  and  thin  sections, 
decolorising  with  such  a  solution  for  fifteen  seconds  is  usually 
sufficient.  Thereafter  the  tissues  are  coloured  by  a  contrast 
stain,  such  as  a  watery  solution  of  methylene-blue  (vide  p.  101). 
The  bacilli  are  also  readily  stained  by  Gram's  method.  Regarding 
the  presence  of  spores  practically  nothing  is  known,  though  some 


FIG.  93. — High -power  view  of  portion  of  leprous  nodule  showing  the 
arrangement  of  the  bacilli  within  the  cells  of  the  granulation  tissue. 

Paraffin  section  ;  stained  with  carbol-fuchsin  and  methylene-blue.      x  1100. 

of  the  unstained  or  stained  points  may  be  of  this  nature.  We 
have,  however,  no  means  of  testing  their  powers  of  resistance. 
Leprosy  bacilli  are  non-motile. 

Position  of  the  Bacilli. — They  occur  in  enormous  numbers 
in  the  leprous  lesions,  especially  in  the  tubercular  form.  In  fact, 
so  numerous  are  they  that  the  granulation  tissue  in  sections, 
properly  stained  as  above,  presents  quite  a  red  colour  under  a  low 
power  of  the  microscope.  The  bacilli  occur  for  the  most  part 
within  the  protoplasm  of  the  round  cells  of  the  granulation  tissue, 
and  are  often  so  numerous  that  the  structure  of  the  cells  is  quite 


272  LEPROSY 

obscured  (Fig.  92).  They  are  often  arranged  in  bundles  which 
contain  several  bacilli  lying  parallel  to  one  another,  though  the 
bundles  lie  in  various  directions  (Fig.  93).  The  appearance  thus 
presented  by  the  cells  filled  with  bacilli  is  very  characteristic. 
Bacilli  are  also  found  free  in  the  lymphatic  spaces,  but  the  greater 
number  are  undoubtedly  contained  within  the  cells.  They  are 
also  found  in  spindle-shaped  connective-tissue  cells,  in  endothelial 
cells,  and  in  the  walls  of  blood  vessels.  They  are  for  the  most 
part  confined  to  the  connective  tissue,  but  a  few  may  be 
seen  in  the  hair  follicles  and  glands  of  the  skin.  Occasionally 
one  or  two  may  be  found  in  the  surface  epithelium,  where  they 
probably  have  been  carried  by  leucocytes,  but  this  position  is,  on 
the  whole,  exceptional.  They  also  occur  in  large  numbers  in  the 
lymphatic  glands  associated  with  the  affected  parts.  In  the 
internal  organs — liver,  spleen,  etc.,  when  leprous  lesions  are 
present,  the  bacilli  are  also  found  though  in  relatively  smaller 
numbers.  In  the  nerves  in  the  anaesthetic  form  they  are  com- 
paratively few,  and  in  the  sclerosed  parts  it  may  be  impossible  to 
find  any.  There  are  few  also  in  the  skin  patches  referred  to 
above  as  occurring  in  this  form  of  the  disease. 

Their  spread  is  chiefly  by  the  lymphatics,  though  distribution 
by  the  blood  stream  also  occurs.  They  have  been  said  to  be 
found  in  the  blood  during  the  presence  of  fever  and  the  eruption 
of  fresh  nodules,  and  they  have  also  been  observed  in  the  blood 
vessels  post  mortem,  chiefly  contained  within  leucocytes.  Recent 
observations  (e.g.  those  of  Doutrelepont  and  Wolters)  show  that 
the  bacilli  may  be  more  widely  spread  throughout  the  body  than 
was  formerly  supposed.  A  few  may  be  detected  in  some  cases 
in  various  organs  which  show  no  structural  change,  especially  in 
their  capillaries.  The  brain  and  spinal  cord  are  almost  exempt, 
but  in  some  cases  bacilli  have  been  found  even  within  nerve 
cells. 

Relations  to  the  Disease. — Attempts  to  cultivate  the  leprosy 
bacilli  outside  the  body  have  so  far 'been  unsuccessful.  From 
time  to  time  announcements  of  successful  cultivations  have  been 
made,  but  one  after  another  has  proved  to  be  erroneous.  A 
similar  statement  may  be  made  with  regard  to  experiments  on 
animals.  If  a  piece  of  leprous  tissue  be  introduced  subcutaneously 
in  an  animal,  such  as  the  rabbit,  a  certain  amount  of  induration 
may  take  place  around  it,  and  the  bacilli  may  be  found  unchanged 
in  appearance  weeks  or  even  months  afterwards,  but  no  multi- 
plication of  the  organisms  occurs.  The  only  exception  to  this 
statement  is  afforded  by  the  experiments  of  Melcher  andOrthmann, 
who  inoculated  the  anterior  chamber  of  the  eye  of  rabbits  with 


RELATIONS   TO   THE   DISEASE  273 

leprous  material,  the  inoculation  being  followed  by  an  extensive 
growth  of  nodules  in  the  lungs  and  internal  organs,  which  they 
affirmed  contained  leprosy  bacilli.  It  has  been  questioned, 
however,  by  several  authorities  whether  the  organisms  in  the 
nodules  were  really  leprosy  bacilli,  and  up  to  the  present  we 
cannot  say  that  there  is  any  satisfactory  proof  that  the  disease  can 
be  transmitted  to  any  of  the  lower  animals.  Diphtheroid  bacilli 
of  more  than  one  variety  have  been  cultivated  from  the  blood 
and  tissues  of  leprous  patients  by  Babes  and  others.  Their 
presence  would  appear  to  be  by  no  means  infrequent,  but  it  is 
not  possible  to  say  at  present  what  their  significance  is. 

It  is  interesting  to  note  that  a  disease  occurs  under  natural 
conditions  in  rats  which  presents  many  points  of  close  similarity 
to  leprosy.  It  has  been  observed  in  Russia,  Germany,  and 
England,  and  an  excellent  description  has  recently  been  given  by 
Dean.  In  this  affection  there  are  lesions  in  the  skin  which 
resemble  those  in  leprosy,  and  the  cells  contain  enormous 
numbers  of  an  acid -fast  bacillus.  The  disease  can  be  trans- 
mitted to  rats  by  inoculation  with  the  tissue  juices  containing 
the  bacilli,  but  riot  to  animals  of  other  species.  All  attempts  to 
cultivate  the  characteristic  organism  outside  the  body  have 
failed,  but  Dean  has  obtained  a  diphtheroid  bacillus — a  result  of 
interest  in  relation  to  what  has  been  found  in  leprosy.  Whether 
this  disease  has  any  relation  to  leprosy  in  the  human  subject  is 
very  doubtful,  but  the  facts  which  have  been  ascertained  may 
prove  of  high  importance  in  connection  with  the  pathology  of 
the  latter  disease. 

It  would  also  appear  that  the  disease  is  not  readily  inoculable 
in  the  human  subject.  In  a  well-known  case  described  by  Arning, 
a  criminal  in  the  Sandwich  Islands  was  inoculated  in  several  parts 
of  the  body  with  leprosy  tissue.  Two  or  three  years  later,  well- 
marked  tubercular  leprosy  appeared  and  led  to  a  fatal  result. 
This  experiment,  however,  is  open  to  the  objection  that  the 
individual  before  inoculation  had  been  exposed  to  infection  in  a 
natural  way,  having  been  frequently  in  contact  with  lepers.  In 
other  cases,  inoculation  experiments  on  healthy  subjects  and 
inoculations  in  other  parts  of  leprous  individuals  have  given 
negative  results.  It  has  been  supposed  by  some  that  the  failure 
to  obtain  cultures  and  to  reproduce  the  disease  experimentally 
may  be  partly  due  to  the  bacilli  in  the  tissues  being  dead.  That 
many  of  the  leprous  bacilli  are  in  a  dead  condition  is  quite 
possible,  in  view  of  the  long  period  during  which  dead  tubercle 
bacilli  introduced  into  the  tissues  of  animals  retain  their  form  and 
staining  reaction.  There  is  also  the  fact  that  from  time  to  time 
18 


274  LEPROSY 

in  leprous  subjects  there  occur  febrile  attacks,  which  are  followed 
by  a  fresh  outbreak  of  nodules,  and  it  would  appear  that 
especially  at  these  times  multiplication  of  the  bacilli  takes  place 
more  actively. 

The  facts  stated  with  regard  to  cultivation  and  inoculation 
experiments  go  to  distinguish  the  leprosy  bacillus  all  the  more 
strongly  from  other  organisms.  Some  have  supposed  that 
leprosy  is  a  form  of  tubercle,  or  tubercle  modified  in  some  way, 
but  for  this  there  appears  to  us  to  be  no  evidence.  Both  from 
the  pathological  and  from  the  bacteriological  point  of  view  the 
diseases  are  distinct.  It  should  also  be  mentioned  that  tubercle 
is  a  not  uncommon  complication  in  leprous  subjects,  in  which 
case  it  presents  the  ordinary  characters. 

The  mode  by  which  leprosy  is  transmitted  has  been  the 
subject  of  great  controversy,  and  is  one  on  which  authorities  still 
hold  opposite  opinions.  Some  consider  that  it  is  a  hereditary 
disease,  or  at  least  that  it  is  transmitted  from  a  parent  to  the 
offspring ;  others  again  that  it  is  transmitted  by  direct  contact. 
There  appears  to  be  no  doubt,  however,  that  on  the  one  hand 
leprous  subjects  may  bear  children  free  from  leprosy,  and  that  on 
the  other  hand,  healthy  individuals  entering  a  leprous  district  may 
contract  the  disease,  though  this  rarely  occurs.  Of  the  latter 
occurrence  there  is  the  well-known  instance  of  Father  Damien, 
who  contracted  leprosy  after  going  to  the  Sandwich  Islands.  In 
view  of  all  the  facts  there  can  be  little  doubt  that  leprosy  in 
certain  conditions  may  be  transmitted  by  direct  contact,  though 
its  contagiousness  is  not  of  a  high  order. 

Methods  of  Diagnosis. — Film  preparations  should  be  made 
with  the  discharge  from  any  ulcerated  nodule  which  may  be 
present,  or  from  the  scraping  of  a  portion  of  excised  tissue,  and 
should  be  stained  as  above  described.  The  presence  of  large 
numbers  of  bacilli  situated  within  the  cells  and  giving  the  staining 
reaction  of  leprosy  bacilli,  is  conclusive.  It  is  more  satisfactory, 
however,  to  make  microscopic  sections  through  a  portion  of  the 
excised  tissue,  when  the  structure  of  the  nodule  and  the  arrange- 
ment of  the  bacilli  can  be  readily  studied.  The  points  of 
difference  between  leprosy  and  tubercle  have  already  been  stated, 
and  in  most  cases  there  is  really  no  difficulty  in  distinguishing 
the  two  conditions. 


CHAPTER  XL 

GLANDERS   AND   RHINOSCLEROMA. 

GLANDERS. 

THE  bacillus  of  glanders  (bacillus  mallei ;  Fr.,  bacille  de  la 
morve  •  Ger.,  Rotzbacillus)  was  discovered  by  Loffler  and  Schutz, 
the  announcement  of  this  discovery  being  made  towards  the  end 
of  1882.  They  not  only  obtained  pure  cultures  of  this  organism 
from  the  tissues  in  the  disease,  but  by  experiments  on  horses 
and  other  animals  conclusively  established  its  causal  relationship. 
These  have  been  fully  confirmed.  The  same  organism  has  also 
been  cultivated  from  the  disease  in  the  human  subject,  first  by 
Weichselbaum  in  1885,  who  obtained  it  from  the  pustules  in  a 
case  of  acute  glanders  in  a  woman,  and  by  inoculation  of  animals 
obtained  results  similar  to  those  of  Loffler  and  Schutz. 

Within  recent  years  a  substance,  mallein,  has  been  obtained 
from  the  cultures  of  the  glanders  bacillus  by  a  method  similar 
to  that  by  which  tuberculin  was  prepared,  and  has  been  found 
to  produce  corresponding  effects  in  animals  suffering  from 
glanders  to  those  produced  by  tuberculin  in  tuberculous  animals. 

The  Natural  Disease. — Glanders  chiefly  affects  the  equine 
species — horses,  mules,  and  asses.  Horned  cattle,  on  the  other 
hand,  are  quite  immune,  whilst  goats  and  sheep  occupy  an  inter- 
mediate position,  the  former  being  rather  more  susceptible  and 
occasionally  suffering  from  the  natural  disease.  It  also  occurs 
in  some  of  the  carnivora — cats,  lions,  and  tigers  in  menageries, 
which  animals  are  infected  from  the  carcases  of  animals  affected 
with  the  disease.  Many  of  the  small  rodents  are  highly 
susceptible  to  inoculation  (vide  infra). 

Glanders  is  also  found  in  man  as  the  result  of  direct  inocula- 
tion on  some  wound  of  the  skin  or  other  part  by  means  of  the 
discharges  or  diseased  tissues  of  an  animal  affected,  and  hence  is 
commonest  amongst  grooms  and  others  whose  work  brings  them 

275 


276  GLANDERS 

into  contact  with  horses ;  even  amongst  them  it  is  a  comparatively 
rare  disease. 

In  horses  the  lesions  are  of  two  types,  to  which  the  names  "glanders  " 
proper  and  "farcy"  have  been  given,  though  both  may  exist  together. 
In  glanders  proper  the  septum  nasi  and  adjacent  parts  are  chiefly  affected, 
there  occurring  in  the  mucous  membrane  nodules  which  are  at  first  firm 
and  of  somewhat  translucent  grey  appearance.  The  growth  of  these  is 
attended  usually  by  inflammatory  swelling  and  profuse  catarrhal  dis- 
charge. Afterwards  the  nodules  soften  in  the  centre,  break  down,  and 
give  rise  to  irregular  ulcerations.  Similar  lesions,  though  in  less  degree, 
may  be  found  in  the  respiratory  passages.  Associated  with  these  lesions 
there  is  usually  implication  of  the  lymphatic  glands  in  the  neck,  media- 
stinum, etc.  ;  and  there  may  be  in  the  lungs,  spleen,  liver,  etc. ,  nodules 
of  the  size  of  a  pea  or  larger,  of  greyish  or  yellow  tint,  firm  or  somewhat 
softened  in  the  centre,  and  often  surrounded  by  a  congested  zone.  The 
term  "farcy"  is  applied  to  the  affection  of  the  superficial  lymphatic 
vessels  and  glands,  which  is  specially  seen  where  infection  takes  place 
through  an  abrasion  of  the  skin,  such  as  is  often  produced  by  the  rubbing 
of  the  harness.  The  lymphatic  vessels  become  irregularly  thickened,  so 
as  to  appear  like  knotted  cords,  and  the  associated  lymphatic  glands 
become  enlarged  and  firm,  though  suppurative  softening  usually  follows, 
and  there  may  be  ulceration.  These  thickenings  are  often  spoken  of  as 
"  farcy  buds  "  and  "  farcy  pipes."  In  farcy,  also,  secondary  nodules  may 
occur  in  internal  organs  and  the  nasal  mucous  membrane.  The  disease 
is  often  present  in  a  "latent  form,"  and  its  presence  can  only  be  detected 
by  the  mallein  test  (vide  infra}.  In  the  ass  the  disease  runs  a  more 
acute  course  than  in  the  horse. 

In  man  the  disease  is  met  with  in  two  forms,  an  acute  and  a 
chronic  ;  though  intermediate  forms  also  occur,  and  chronic  cases 
may  take  on  the  characters  of  the  acute  disease.  The  site  of 
inoculation  is  usually  on  the  hand  or  arm,  by  means  of  some 
scratch  or  abrasion,  or  possibly  along  a  hair  follicle,  sometimes  on 
the  face,  and  occasionally  on  the  mucous  membrane  of  the  mouth, 
nose,  or  eye.  In  the  acute  form  there  appears  at  the  site  of 
inoculation  inflammatory  swelling,  attended  usually  with  spread- 
ing redness,  and  the  lymphatics  in  relation  to  the  part  also  become 
inflamed,  the  appearances  being  those  of  a  "  poisoned  wound." 
These  local  changes  are  soon  followed  by  marked  constitutional 
disturbance,  and  by  an  eruption  on  the  surface  of  the  body,  at 
first  papular  and  afterwards  pustular,  and  later  there  may  form  in 
the  subcutaneous  tissue  and  muscles  larger  masses  which  soften 
and. suppurate,  the  pus  being  often  mixed  with  blood  ;  suppuration 
may  occur  also  in  the  joints.  In  some  cases  the  nasal  mucous 
membrane  may  be  secondarily  infected,  and  thence  inflammatory 
swelling  may  spread  to  the  tissues  of  the  face ;  in  others  it 
remains  free.  The  patient  usually  dies  in  two  or  three  weeks, 
sometimes  sooner,  with  the  symptoms  of  rapid  pyaemia.  In 


THE   GLANDERS   BACILLUS 


277 


addition  to  the  lesions  mentioned  there  may  be  foci,  usually 
suppurative,  in  the  lungs  (attended  often  with  pneumonic  con- 
solidation), in  the  spleen,  liver,  bone-marrow,  salivary  glands,  etc. 
In  the  chronic  form  the  local  lesion  results  in  the  formation  of  an 
irregular  ulcer  with  thickened  margins  and  sanious,  often  foul, 
discharge.  The  ulceration  spreads  deeply  as  well  as  superficially, 
and  the  thickened  lymphatics  also  have  a  great  tendency  to 
ulcerate,  though  the  lymphatic  system  is  not  so  prominently 
affected  as  in  the  horse.  Deposits  may  form  in  the  subcutaneous 
tissue  and  muscles,  and  the  mucous  membrane  may  become 
affected.  The  disease  may 
run  a  very  chronic  course, 
lasting  for  months,  and 
recovery  may  occur, 
though,  on  the  other  hand, 
the  disease  may  take  on 
a  more  acute  character 
and  rapidly  become  fatal. 

The  Glanders  Bacil- 
lus. —  Microscopical 
Characters.  —  The  glan- 
ders bacilli  are  minute 
rods,  straight  or  slightly 
curved,  with  rounded 
ends,  and  about  the  same 
length  as  tubercle  bacilli, 
but  distinctly  thicker  FlG  94._Glanders  bacim  amongst  broken- 
(Fig.  94 ).  They  show,  down  cells.  Film  preparation  from  a 

however,          considerable          glanders  nodule  in  a  guinea-pig, 
variations  in  size  and  in    Stained  with  weak  carbol-fuchsiu.      x  1000. 
appearance,  and  their  pro- 
toplasm is  often  broken  up  into  a  number  of  deeply-stained 
portions  with  unstained  intervals  between.     These  characters  are 
seen  both  in  the  tissues  and  in  cultures,  but,  as  in  the  case  of 
many  organisms,  irregularities  in  form  and  size  are  more  pro- 
nounced in  cultures  (Fig  95) ;  short  filamentous  forms  8  to  12  p  in 
length  are  sometimes  met  with,  but  these  are  on  the  whole  rare. 
The  organism  is  non-motile. 

In  the  tissues  the  bacilli  usually  occur  irregularly  scattered 
amongst  the  cellular  elements  ;  a  few  may  be  contained  within 
leucocytes  and  connective-tissue  corpuscles,  but  the  position  of 
most  is  extracellular.  They  are  most  abundant  in  the  acute  lesions, 
in  which  they  may  be  found  in  considerable  numbers  ;  but  in  the 
chronic  nodules,  especially  when  softening  has  taken  place,  they 


278 


GLANDERS 


are  few  in  number,  and  it  may  be  impossible  to  find  any  in 
sections.  They  have  less  powers  of  persistence,  and  disappear  in 
the  tissues  much  more  quickly  than  tubercle  bacilli. 

There  has  been  dispute  as  to  whether  or  not  they  contain 
spores.     Some  consider  certain  of  the  unstained  portions  to  be 

of  that  nature,  and  it  has 
been  'claimed  that  these 
can  be  stained  by  the 
method  for  staining  spores 
(Rosen thai).  But  it  is 
very  doubtful  that  such  is 
the  case ;  the  appearances 
correspond  rather  with 
mere  breaks  in  the  proto- 
plasm, such  as  are  met 
with  in  many  other  bacilli 
which  do  not  contain 
spores,  and  the  compara- 
tively low  powers  of  resist- 
ance of  glanders  bacilli 
containing  these  so-called 


FIG.    95. — Glanders    bacilli, 
culture  on  glycerin  agar. 
carbol-fuchsinand  partially  decolorised  to 
show  segmentation  of  protoplasm,   x  1000. 


spores,  is  strongly  against 
from    a    pure    ,  f  .    ,    .         £  ,  ?  ' 
Stained  with   their  being  of  that  nature. 


The  power  of  resistance  is 
after  all  the  important 
practical  point. 

Staining. — The  glanders  bacillus  differs  widely  from  the 
tubercle  bacillus  in  its  staining  reactions.  It  stains  with  simple 
watery  solutions  of  the  basic  stains,  but  somewhat  faintly  (better 
when  an  alkali  or  a  mordant,  such  as  carbolic  acid,  is  added), 
and  even  when  deeply  stained  it  readily  loses  the  colour  when  a 
decolorising  agent  such  as  alcohol  is  applied.  We  have  obtained 
the  best  results  by  carbol-thionin-blue  (p.  98),  and  we  prefer  to 
dehydrate  by  the  aniline-oil  method.  In  film  preparations  of 
fresh  glanders  nodules  the  bacilli  can  be  readily  found  by  staining 
with  any  of  the  ordinary  combinations,  e.g.  carbol-thionin-blue 
or  weak  carbol-fuchsin.  By  using  a  stain  of  suitable  strength 
no  decolorising  agent  is  necessary,  the  film  being  simply  washed 
in  water,  dried  and  mounted.  M'Fadyean  recommends  that 
after  sections  have  been  stained  in  Lomer's  methylene  blue  and 
slightly  decolorised  in  weak  acetic  acid,  they  should  be  treated 
for  fifteen  minutes  with  a  saturated  solution  of  tannic  acid ; 
thereafter  they  are  washed  thoroughly  in  water,  and  as  a  contrast 
stain  a  1  per  cent  solution  of  acid  fuchsin  may  be  applied  for 


CULTIVATION   OF   GLANDERS   BACILLUS     279 

half  a  minute ;  they  are  then  dehydrated,  cleared,  and  mounted. 
Gram's  method  is  quite  inapplicable,  the  glanders  bacilli  rapidly 
losing  the  stain  in  the  process. 

Cultivation. — (For  the  methods  of  separation  vide  infra.) 
The  glanders  bacillus  grows  readily  on  most  of  the  ordinary 
media,  but  a  somewhat  high  temperature  is  necessary,  growth 
taking  place  most  rapidly  at  35°  to  37°  C.  Though  a  certain 
amount  of  growth  occurs  down  to  21°  C.,  a  temperature  above 
25°  C.  is  always  desirable. 

On  agar  and  glycerin  agar  in  stroke  cultures  growth  appears 
along  the  line  as  a  uniform  streak  of  greyish- white  colour  and 
somewhat  transparent  appearance,  with  moist-looking  surface, 
and  when  touched  with  a  needle  is  found  to  be  of  rather  slimy 
consistence.  Later  it  spreads  laterally  for  some  distance,  and 
the  layer  becomes  of  slightly  brownish  tint.  On  serum  the 
growth  is  somewhat  similar  but  more  transparent,  the  separate 
colonies  being  in  the  form  of  round  and  almost  clear  drops.  In 
sub-cultures  on  these  media  at  the  body  temperature  growth  is 
visible  within  twenty-four  hours,  but  when  fresh  cultures  are 
made  from  the  tissues  it  may  not  be  visible  till  the  second  day. 
Serum  or  potato,  however,  is  much  more  suitable  for  cultivating 
from  the  tissues  than  the  agar  media ;  on  the  latter  it  is  some- 
times difficult  to  obtain  growth. 

In  broth,  growth  forms  at  first  a  uniform  turbidity,  but  soon 
settles  to  the  bottom,  and  after  a  few  days  forms  a  pretty  thick 
fiocculent  deposit  of  slimy  and  somewhat  tenacious  consistence. 

On  potato  at  30°  to  37°  C.  the  glanders  bacillus  flourishes  well 
and  produces  a  characteristic  appearance ;  incubation  at  a  high 
temperature,  however,  being  necessary.  Growth  proceeds  rapidly, 
and  on  the  third  day  has  usually  formed  a  transparent  layer  of 
slightly  yellowish  tint,  like  clear  honey  in  appearance.  On  sub- 
sequent days,  the  growth  still  extends  and  becomes  darker  in  colour 
and  more  opaque,  till  about  the  eighth  day  it  has  a  reddish-brown 
or  chocolate  tint,  while  the  potato  at  the  margin  of  the  growth 
often  shows  a  greenish-yellow  staining.  The  characters  of  the 
growth  on  potato  along  with  the  microscopical  appearances  are 
quite  sufficient  to  distinguish  the  glanders  bacillus  from  every 
other  known  organism  (sometimes  the  cholera  organism  and  the 
b.  pyocyaneus  produce  a  somewhat  similar  appearance,  but  they 
can  be  readily  distinguished  by  their  other  characters).  Potato 
is  also  a  suitable  medium  for  starting  cultures  from  the  tissues ; 
in  this  case  minute  transparent  colonies  become  visible  on  the 
third  day  and  afterwards  present  the  appearances  just  described. 

Powers  of  Resistance. — The  glanders  bacillus  is  not  killed  at 


280  GLANDERS 

once  by  drying,  but  usually  loses  its  vitality  after  fourteen  days 
in  the  dry  condition,  though  sometimes  it  lives  longer.  It  is  not 
quickly  destroyed  by  putrefaction,  having  been  found  to  be  still 
active  after  remaining  two  or  three  weeks  in  putrefying  fluids. 
In  cultures  the  bacilli  retain  their  vitality  for  three  or  four 
months,  if,  after  growth  has  taken  place,  they  are  kept  at  the 
temperature  of  the  room;  on  the  other  hand,  they  are  often 
found  to  be  dead  at  the  end  of  two  weeks  when  kept  constantly 
at  the  body  temperature.  They  have  comparatively  feeble 
resistance  to  heat  and  antiseptics.  Lomer  found  that  they  were 
killed  in  ten  minutes  in  a  fluid  kept  at  55°  C.,  and  in  from  two 
to  three  minutes  by  a  5  per  cent  solution  of  carbolic  acid. 
Boiling  water  and  the  ordinarily  used  antiseptics  are  very 
rapid  and  efficient  disinfectants. 

We  may  summarise  the  characters  of  the  glanders  bacillus  by 
saying  that  in  its  morphological  characters  it  resembles  some- 
what the  tubercle  bacillus,  but  is  thicker,  and  differs  widely 
from  it  in  its  staining  reactions.  For  its  cultivation  the  higher 
temperatures  are  necessary,  and  the  growth  on  potato  presents 
most  characteristic  features. 

Experimental  Inoculation. — In  horses  subcutaneous  injection 
of  the  glanders  bacillus  in  pure  culture  reproduces  all  the 
important  features  of  the  disease.  This  fact  was  established  at 
a  comparatively  early  date  by  Loffler  and  Schutz,  who,  after  one 
doubtful  experiment,  successfully  inoculated  two  horses  in  this 
way,  the  cultures  used  having  been  grown  for  several  generations 
outside  the  body.  In  a  few  days  swellings  formed  at  the  sites 
of  inoculation,  and  later  broke  down  into  unhealthy -looking 
ulcers.  One  of  the  animals  died ;  after  a  few  weeks  the  other, 
showing  symptoms  of  cachexia,  was  killed.  In  both  animals,  in 
addition  to  ulcerations  on  the  surface  with  involvement  of  the 
lymphatics,  there  were  found,  post  mortem,  nodules  in  the  lungs, 
softened  deposits  in  the  muscles,  and  also  affection  of  the  nasal 
mucous  membrane,— nodules,  and  irregular  ulcerations.  The 
ass  is  even  more  susceptible  than  the  horse,  the  disease  in  the 
former  running  a  more  rapid  course,  but  with  similar  lesions. 
The  ass  can  be  readily  infected  by  simple  scarification  and 
inoculation  with  glanders  secretion,  etc.  (Nocard). 

Of  small  animals,  field-mice  and  guinea-pigs  are  the  most 
susceptible.  Strangely  enough,  house -mice  and  white  mice 
enjoy  an  almost  complete  immunity.  In  field-mice  subcutaneous 
inoculation  is  followed  by  a  very  rapid  disease,  usually  leading 
to  death  within  eight  days,  the  organisms  becoming  generalised 
and  producing  numerous  minute  nodules,  especially  in  the  spleen, 


ACTION   ON   THE   TISSUES  281 

lungs,  and  liver.  In  the  guinea-pig  the  disease  is  less  acute, 
though  secondary  nodules  in  internal  organs  are  usually  present 
in  considerable  numbers.  At  the  site  of  inoculation  an  inflam- 
matory swelling  forms,  which  soon  softens  and  breaks  down, 
leading  to  the  formation  of  an  irregular  crateriform  ulcer  with 
indurated  margins.  The  lymphatic  vessels  become  infiltrated, 
and  the  corresponding  lymphatic  glands  become  enlarged  to  the 
size  of  peas  or  small  nuts,  softened,  and  semi-purulent.  The 
animal  sometimes  dies  in  two  or  three  weeks,  sometimes  not  for 
a  longer  period.  Secondary  nodules,  in  varying  numbers  in 
different  cases,  may  be  present  in  the  spleen,  lungs,  bones,  nasal 
mucous  membrane,  testicles,  ovaries,  etc.  ;  in  some  cases  a  few 
nodules  are  found  in  the  spleen  alone.  Intraperitoneal  injection 
in  the  male  guinea-pig  is  followed,  as  pointed  out  by  Straus,  by 
a  very  rapid  and  semi-purulent  affection  of  the  tunica  vaginalis, 
shown  during  life  by  great  swelling  and  redness  of  the  testicles, 
which  changes  may  be  noticeable  in  two  or  three  days.  By  this 
method  there  occur  also  numerous  small  nodules  on  the  surface 
of  the  peritoneum.  Rabbits  are  less  susceptible  than  guinea- 
pigs,  and  the  effect  of  subcutaneous  inoculation  is  somewhat 
uncertain.  Accidental  inoculation  of  the  human  subject  with 
pure  cultures  of  the  bacillus  has  in  more  than  one  instance  been 
followed  by  the  acute  form  of  the  disease  and  a  fatal  result. 

Mayer  has  found  that  when  the  glanders  bacillus  is  injected  along 
with  melted  butter  into  the  peritoneum  of  a  guinea-pig,  it  shows 
filamentous,  branching,  and  club  -  shaped  forms  ;  in  other  words,  it 
presents  the  characters  of  a  streptothrix.  Lubarsch,  on  the  other  hand, 
in  a  comparative  study  of  the  results  of  inoculation  with  acid-fast  and 
other  bacilli,  found  none  of  the  above  characters  in  the  case  of  the 
glanders  bacillus  (cf.  Tubercle). 

Action  on  the  Tissues. — From  the  above  facts  it  will  be  seen 
that  in  many  respects  glanders  presents  an  analogy  to  tubercle  as 
regards  the  general  characters  of  the  lesions  and  the  mode  of 
spread.  When  the  tissue  changes  in  the  two  diseases  are 
compared,  certain  differences  are  found.  The  glanders  bacillus 
causes  a  more  rapid  and  more  marked  inflammatory  reaction. 
There  is  more  leucocytic  infiltration  and  less  proliferative  change 
which  might  lead  to  the  formation  of  epithelioid  cells.  Thus 
the  centre  of  an  early  glanders  nodule  shows  a  dense  aggregation 
of  leucocytes,  most  of  which  are  polymorpho-nuclear,  whilst  in 
the  central  parts  many  show  fragmentation  of  nuclei  with  the 
formation  of  a  deeply  staining  granular  detritus.  And  further, 
the  inflammatory  change  may  be  followed  by  suppurative 
softening  of  the  tissue,  especially  in  certain  situations,  such  as 


282  GLANDERS 

the  subcutaneous  tissue  and  lymphatic  glands.  The  nodules, 
therefore,  in  glanders,  as  Baumgarten  puts  it,  occupy  an 
intermediate  position  between  miliary  abscesses  and  tubercles. 
The  diffuse  coagulative  necrosis  and  caseation  which  are  so 
common  in  tubercle  do  not  occur  to  the  same  degree  in  glanders, 
and  typical  giant  cells  are  not  formed.  The  nodules  in  the  lungs 
show  leucocytic  infiltration  and  thickening  of  the  alveolar  walls, 
whilst  the  vesicles  are  filled  with  catarrhal  cells ;  there  may  also 
be  fibrinous  exudation,  whilst  at  the  periphery  of  the  nodules  con- 
nective-tissue growth  is  present  in  proportion  to  their  age.  The 
tendency  to  spread  by  the  lymphatics  is  always  a  well-marked 
feature,  and  when  the  bacilli  gain  entrance  to  the  blood-stream, 
they  soon  settle  in  the  various  tissues  and  organs.  Accordingly, 
even  in  acute  cases  it  is  usually  quite  impossible  to  detect  the 
bacilli  in  the  circulating  blood,  though  sometimes  they  have  been 
found.  It  is  an  interesting  fact,  shown  by  observations  of  the 
disease  both  in  the  human  subject  and  in  the  horse,  as  well  as 
by  experiments  on  guinea-pigs,  that  the  mucous  membrane  of  the 
nose  may  become  infected  by  means  of  the  blood-stream — another 
example  of  the  tendency  of  organisms  to  settle  in  special  sites. 

Mode  of  Spread. — Glanders  usually  spreads  from  a  diseased 
animal  by  direct  contagion  with  the  discharge  from  the  nose  or 
from  the  sores,  etc.  So  far  as  infection  of  the  human  subject 
goes,  no  other  mode  is  known.  There  is  no  evidence  that  the 
disease  is  produced  in  man  by  inhalation  of  the  bacilli  in  the 
dried  condition.  Some  authorities  consider  that  pulmonary 
glanders  may  be  produced  in  this  way  in  the  horse,  whilst  others 
maintain  that  in  all  cases  there  is  first  a  lesion  of  the  nasal 
mucous  membrane  or  of  the  skin  surface,  and  that  the  lung  is 
affected  secondarily.  Babes,  however,  found  that  the  disease 
could  be  readily  produced  in  susceptible  animals  by  exposing 
them  to  an  atmosphere  in  which  cultures  of  the  bacillus  had 
been  pulverised.  He  also  found  that  inunction  of  the  skin 
with  vaseline  containing  the  bacilli  might  produce  the  disease, 
the  bacilli  in  this  case  entering  along  the  hair  follicles. 

Agglutination  of  Glanders  Bacilli. — Shortly  after  the  discovery  of 
agglutination  in  typhoid  fever,  M'Fadyean  showed  that  the  serum  of 
glandered  horses  possessed  the  power  of  agglutinating  glanders  bacilli. 
His  later  observations  show  that  in  the  great  majority  of  cases  of  glanders 
a  1  in  50  dilution  of  the  serum  produces  marked  agglutination  in  a  few 
minutes,  whilst  in  the  great  majority  of  non-glandered  animals  no  effect 
is  produced  under  these  conditions.  The  test  performed  in  the  ordinary 
way  is,  however,  not  absolutely  reliable,  as  exceptions  occasionally 
occur  in  both  directions,  i.e.  negative  results  by  glandered  anmials  and 
positive  results  by  non-glandered  animals.  He  finds  that  a  more  delicate 


METHODS   OF   EXAMINATION  283 

and  reliable  method  is  to  grow  the  bacillus  in  bouillon  containing  a  small 
proportion  of  the  serum  to  be  tested.  In  this  way  he  has  obtained  a 
distinct  sedimenting  reaction  with  a  serum  which  did  not  agglutinate  at 
all  distinctly  in  the  ordinary  method.  Further  observations  are  still 
required  to  determine  the  precise  value  of  the  test. 

Mallein  and  its  Preparation. — Mallein  is  obtained  from  cultures  of  the 
glanders  bacillus  grown  for  a  suitable  length  of  time,  and,  like  tuber- 
culin, is  really  a  mixture  comprising  (1)  substances  in  the  bodies  of  the 
bacilli  and  (2)  their  soluble  products,  not  destroyed  by  heat,  along  with 
substances  derived  from  the  medium  of  growth.  It  was  at  first  obtained 
from  cultures  on  solid  media  by  extracting  with  glycerin  or  water,  but  is 
now  usually  prepared  from  cultures  in  glycerin  bouillon.  Such  a  culture, 
after  being  allowed  to  grow  for  three  or  four  weeks,  is  sterilised  by  heat 
either  in  the  autoclave  at  115°  C.  or  by  steaming  at  100°  C.  on  successive 
days.  It  is  then  filtered  through  a  Chamberland  filter.  The  filtrate 
constitutes  fluid  mallein.  Usually  a  little  carbolic  acid  ('5  per  cent)  is 
added  to  prevent  it  from  decomposing.  Of  such  mallein  1  c.c.  is  usually 
the  dose  for  a  horse  (M'Fadyean).  Foth  has  prepared  a  dry  form  of 
malleiu  by  throwing  the  filtrate  of  a  broth  culture,  evaporated  to  one- 
tenth  of  its  bulk,  into  twenty-five  or  thirty  times  its  volume  of  alcohol. 
A  white  precipitate  is  formed,  which  is  dried  over  calcium  chloride  and 
then  under  an  air-pump.  A  dose  of  this  dry  mallein  is  '05  to  "07  grm. 

The  use  of  Mallein  as  a  means  of  Diagnosis. — In  using  mallein  for  the 
diagnosis  of  glanders,  the  temperature  of  the  animal  ought  to  be  observed 
for  some  hours  beforehand,  and,  after  subcutaneous  injection  of  a  suitable 
dose,  it  is  taken  at  definite  intervals, — according  to  M'Fadyean  at  the 
sixth,  tenth,  fourteenth,  and  eighteenth  hours  afterwards,  and  on  the 
next  day.  Here  both  the  local  reaction  and  the  temperature  are  of 
importance.  In  a  glandered  animal,  at  the  site  of  inoculation  there  is  a 
somewhat  painful  local  swelling,  which  reaches  a  diameter  of  five  inches 
at  least,  the  maximum  size  not  being  attained  until  twenty-four  hours 
afterwards.  The  temperature  rises  1'5°  to  2°  C.,  or  more,  the  maximum 
generally  occurring  in  from  eight  to  sixteen  hours.  If  the  temperature 
never  rises  as  much  as  1  '5°,  the  reaction  is  considered  doubtful.  In  the 
negative  reaction  given  by  an  animal  free  from  glanders,  the  rise  of 
temperature  does  not  usually  exceed  1°,  the  local  swelling  reaches  the 
diameter  of  three  inches  at  most,  and  has  much  diminished  at  the  end 
of  twenty- four  hours.  In  the  case  of  dry  mallein,  local  reaction  is  less 
marked.  Veterinary  authorities  are  practically  unanimous  as  to  the 
great  value  of  the  mallein  test  as  a  means  of  diagnosis. 

Methods  of  Examination. — Microscopic  examination  in  a 
case  of  suspected  glanders  will  at  most  reveal  the  presence  of 
bacilli  corresponding  in  their  characters  to  the  glanders  bacillus. 
An  absolute  diagnosis  cannot  be  made  by  this  method.  Cultures 
may  be  obtained  by  making  successive  strokes  on  blood  serum  or 
on  glycerin  agar  (preferably  the  former),  and  incubating  at  37°  C. 
The  colonies  of  the  glanders  bacillus  do  not  appear  till  two  days 
after.  This  method  often  fails  unless  a  considerable  number  of 
the  glanders  bacilli  are  present.  Another  method  is  to  dilute  the 
secretion  or  pus  with  sterile  water,  to  varying  degrees,  and  then 
to  smear  the  surface  of  potato  with  the  mixture,  the  potatoes 


284  GLANDERS 

being  incubated  at  the  above  temperature.  The  colonies  on 
potatoes  may  not  appear  till  the  third  day.  The  most  certain 
method,  however,  is  by  inoculation  of  a  guinea-pig,  either  by 
subcutaneous  or  intraperitoneal  injection.  By  the  latter  method, 
as  above  described,  lesions  are  much  more  rapidly  produced,  and 
are  more  characteristic.  If,  however,  there  have  been  other 
organisms  present,  the  animal  may  die  of  a  septic  peritonitis, 
though  even  in  such  a  case  the  glanders  bacilli  will  be  found  to 
be  more  numerous  in  the  tunica  vaginalis,  and  may  be  cultivated 
from  this  situation.  It  is  extremely  doubtful  whether  the 
application  of  mallein  to  diagnosis  of  the  disease  in  the  human 
subject  is  justifiable.  There  is  a  certain  risk,  that  it  may  lead 
to  the  lesions  assuming  a  more  acute  character ;  moreover, 
culture  and  inoculation  tests  are  generally  available.  In  the 
case  of  horses,  etc.,  a  diagnosis  will,  however,  be  much  more 
easily  and  rapidly  effected  by  means  of  mallein. 

RHINOSCLEROMA. 

This  disease  is  considered  here  as,  from  the  anatomical 
changes,  it  also  belongs  to  the  group  of  infective  granulomata. 
It  is  characterised  by  the  occurrence  of  chronic  nodular 
thickenings  in  the  skin  or  mucous  membrane  of  the  nose,  or 
in  the  mucous  membrane  of  the  pharynx,  larynx,  or  upper  part 
of  the  trachea.  The  nodules  are  of  considerable  size,  sometimes 
as  large  as  a  pea ;  in  the  earlier  stages  they  are  comparatively 
smooth  on  the  surface,  but  later  they  become  shrunken  and  the 
centre  is  often  retracted.  The  disease  is  scarcely  ever  met  with 
in  this  country,  but  is  of  not  very  uncommon  occurrence  on  the 
Continent,  especially  in  Austria  and  Poland.  In  the  granulation 
tissue  of  the  nodules  there  are  to  be  found  numerous  round  and 
rather  large  cells,  which  have  peculiar  characters  and  are  often 
known  as  the  cells  of  Mikulicz.  Their  protoplasm  contains  a 
collection  of  somewhat  gelatinous  material  which  may  fill  the 
cell  and  push  the  nucleus  to  the  side.  Within  these  cells  there 
is  present  a  characteristic  bacillus,  occurring  in  little  clumps  or 
masses  chiefly  in  the  gelatinous  material.  A  few  bacilli  also  lie 
free  in  the  lymphatic  spaces  around.  This  organism  was  first 
observed  by  Frisch,  and  is  now  known  as  the  bacillus  of 
rhinoscleroma.  The  bacilli  have  the  form  of  short  oval  rods, 
which,  when  lying  separately,  can  be  seen  to  possess  a  distinct 
capsule,  and  which  in  all  their  microscopical  characters  correspond 
closely  with  Friedlander's  pneumobacillus.  They  are  usually 
present  in  the  lesions  in  a  state  of  purity.  It  was  at  first  stated 


RHINOSCLEROMA  285 

that  they  could  be  stained  by  Gram's  method,  but  more  recent 
observations  show  that  like  Friedlander's  organism  they  lose  the 
stain. 

From  the  affected  tissues  this  bacillus  can  be  easily  cultivated 
by  the  ordinary  methods.  In  the  characters  of  its  growth  in 
the  various  culture  media  it  presents  a  close  similarity  to  that 
of  the  pneumobacillus,  as  it  also  does  in  its  fermentative  action 
in  milk  and  sugar-containing  fluids.  The  nail-like  appearance 
of  the  growth  or  gelatin  is  said  to  be  less  distinct,  and  the 
growth  on  potatoes  is  more  transparent  and  may  show  small 
bubbles  of  gas  ;  but  it  is  doubtful  whether  any  distinct  line  of 
difference  can  be  drawn  between  the  two  organisms  so  far  as 
their  microscopical  and  cultural  characters  are  concerned. 

The  evidence  that  the  organisms  described  are  the  cause  of 
this  disease  consists  in  their  constant  presence  and  their  special 
relation  to  the  affected  tissues,  as  already  described.  From 
these  facts  alone  it  would  appear  probable  that  they  are  the 
active  agents  in  the  production  of  the  lesions.  Experimental 
inoculation  has  thrown  little  light  on  the  subject,  though  one 
observer  has  described  the  production  of  nodules  on  the 
conjunctivas  of  guinea-pigs.  The  relation  of  the  rhinoscleroma 
organism  to  that  of  Friedlander  is,  however,  still  a  matter  of 
doubt,  and  the  matter  has  been  further  complicated  by  the  fact 
that  a  bacillus  possessing  closely  similar  characters  has  been 
found  to  be  very  frequently  present  in  ozoena,  and  is  often 
known  as  the  bacillus  ozcence.  The  last-mentioned  organism  is 
said  to  have  more  active  fermentative  powers.  From  what  has 
been  stated  it  will  be  seen  that  a  number  of  organisms  closely 
allied  in  their  morphological  characters,  have  been  found  in  the 
nasal  cavity  in  healthy  or  diseased  conditions.  There  is  no 
doubt  that  rhinoscleroma  is  a  specific  disease  with  well-marked 
characters  and  it  is  quite  possible  that  one  member  of  this  group 
of  organisms  may  be  the  causal  agent,  though  indistinguishable 
from  others  by  culture  tests.  There  is,  however,  a  tendency  on 
the  part  of  recent  investigators  to  consider  the  "bacillus  of 
rhinoscleroma  "  to  be  identical  with  the  pneumobacillus  and  its 
presence  in  the  affected  tissues  to  represent  merely  a  secondary 
invasion.  The  subject  is  one  on  which  more  light  is  still 
required. 


CHAPTER  XII. 

ACTINOMYCOSIS  AND  ALLIED  DISEASES. 

ACTINOMYCOSIS  is  the  most  important  example  of  a  group  of 
diseases  produced  by  streptothrix  organisms.  It  is  related,  by 
the  characters  of  the  pathological  changes,  to  the  diseases  which 
have  been  described.  The  disease  affects  man  in  common  with 
certain  of  the  domestic  animals,  though  it  is  more  frequent  in 
the  latter,  especially  in  oxen,  swine,  and  horses.  The  parasite 
was  first  discovered  in  the  ox  by  Bol  linger,  and  described  by 
him  in  1877,  the  name  actinomyces  or  ray  fungus  being  from  its 
appearance  applied  to  it  by  the  botanist  Harz.  In  1878  Israel 
described  the  parasite  in  the  human  subject,  and  in  the  following 
year  Ponfick  identified  it  as  being  the  same  as  that  found  in  the 
ox.  Since  that  time  a  large  number  of  cases  have  been  observed 
in  the  human  subject,  the  result  of  investigation  being  to  show- 
that  it  affects  man  much  more  frequently  than  was  formerly 
supposed. 

It  is,  however,  to  be  noted  that  the  term  "  actinomyces,"  as 
originally  used,  does  not  represent  one  parasite  but  a  number  of 
closely  allied  species,  as  cultures  obtained  from  various  sources 
have  presented  considerable  differences  ;  and,  further,  it  is  noted 
that  other  distinct  species  of  streptothrix  have  been  cultivated 
from  isolated  cases  of  disease  in  the  human  subject  where  the 
lesions  resembled  more  or  less  closely  those  of  actinomycosis. 
In  one  or  two  instances  the  organism  has  been  found  to  be 
"acid-fast,"  and  there  is  no  doubt  that  the  actinomyces  group  is 
closely  related  through  intermediate  forms  with  the  tubercle 
group  (vide  p.  239). 

Naked-eye  Characters  of  the  Parasite. — The  actinomyces 
grows  in  the  tissues  in  the  form  of  little  round  masses  or  colonies, 
which,  when  fully  developed,  are  easily  visible  to  the  naked  eye, 
the  largest  being  about  the  size  of  a  small  pin's  head,  whilst  all 
sizes  below  this  may  be  found.  When  suppuration  is  present, 

286' 


CHARACTERS   OF   THE   ACTINOMYCES        287 

they  lie  free  in  the  pus ;  when  there  is  no  suppuration,  they  are 
embedded  in  the  granulation  tissue,  but  are  usually  surrounded 
by  a  zone  of  softer  issue.  They  may  be  transparent  or  jelly- 
like,  or  they  may  be  opaque  and  of  various  colours — white, 
yellow,  greenish,  or  almost  black.  The  appearance  depends 
upon  their  age  and  also  upon  their  structure,  the  younger  colonies 
being  more  or  less  transparent,  the  older  ones  being  generally 
opaque.  Their  colour  is  modified  by  the  presence  of  pigment 
and  by  degenerative  change,  which  is  usually  accompanied  by  a 
yellowish  coloration.  They  are  generally  of  soft,  sometimes 
tallow-like,  consistence,  though  sometimes  in  the  ox  they  are 
gritty,  owing  to  the  presence  of  calcareous  deposit.  They  may 
be  readily  found  in  the  pus  by  spreading  it  out  in  a  thin  layer 
on  a  glass  slide  and  holding  it  up  to  the  light.  They  are  some- 
times described  as  being  always  of  a  distinctly  yellow  colour, 
but  this  is  only  occasionally  the  case ;  in  fact,  in  the  human 
subject  they  occur  much  more  frequently  as  small  specks  of 
semi-translucent  appearance,  and  of  greenish-grey  tint. 

Microscopical  Characters. — The  parasite,  which  is  now 
generally  regarded  as  belonging  to  the  streptothrix  group  of  the 
higher  bacteria  (p.  14),  presents  pleomorphous  characters.  In 
the  colonies,  as  they  grow  in  the  tissues,  three  morphological 
elements  may  be  described,  namely,  filaments,  coccus-like  bodies, 
and  clubs. 

1.  The  filaments  are  comparatively  thin,  measuring  about 
•6  /A  in  diameter,  but  they  are  often  of  great  length.  They  are 
composed  of  a  central  protoplasm  enclosed  by  a  sheath.  The 
latter,  which  is  most  easily  made  out  in  the  older  filaments  with 
granular  protoplasm,  occasionally  contains  granules  of  dark 
pigment.  In  the  centre  of  the  colony  the  filaments  interlace 
with  one  another,  and  form  an  irregular  network  which  may  be 
loose  or  dense ;  at  the  periphery  they  are  often  arranged  in  a 
somewhat  radiating  manner,  and  run  outwards  in  a  wavy  or  even 
spiral  course.  They  also  show  true  branching,  a  character 
which  at  once  distinguishes  them  from  the  ordinary  bacteria. 
Between  the  filaments  there  is  a  finely  granular  or  homogeneous 
ground  substance.  Most  of  the  colonies  at  an  early  stage  are 
chiefly  constituted  by  filaments  loosely  arranged ;  but  later,  part 
of  the^growth  may  become  so  dense  that  its  structure  cannot  be 
made  out.  This  dense  part,  starting  excentrically,  may  grow 
round  the  colony  to  form  a  hollow  sphere,  from  the  outer 
surface  of  which  filaments  radiate  for  a  short  distance  (Fig.  96). 
The  filaments  usually  stain  uniformly  in  the  younger  colonies, 
but  some,  especially  in  the  older  colonies,  may  be  segmented  so 


288     ACTINOMYCOSIS   AND   ALLIED   DISEASES 

as  to  give  the  appearance  of  a  chain  of  bacilli  or  of  cocci,  though 
the  sheath  enclosing  them  may  generally  be  distinguished. 
Rod-shaped  and  spherical  forms  may  also  be  seen  lying  free. 

2.  Spores  or  Gonidia. — Like  other  species  of  strep tothrix  the 
actinomyces  when  growing  on  a  culture  medium  shows  on  its 
surface  filaments  growing  upwards  in  the  air,  the  protoplasm  of 
which  becomes  segmented  into  rounded  spores  or  gonidia.  In 
natural  conditions  outside  the  body  these  gonidia  become  free 


FIG.  96. — Actinomycosis  of  human  liver,  showing  a  colony  of  the  parasite 
composed  of  a  felted  mass  of  filaments  surrounded  by  pus. 

Paraffin  section  ;  stained  by  Gram's  method  and  with  safranin.      x  500. 

and  act  as  new  centres  by  growing  out  into  filaments.  They 
have  somewhat  higher  powers  of  resistance  than  the  filaments, 
though  less  than  the  spores  of  most  of  the  lower  bacteria.  An 
exposure  to  75°  C.  for  half  an  hour  is  sufficient  to  kill  most 
streptothrices  or  their  spores ;  cultures  containing  spores  can 
resist  a  temperature  from  five  to  ten  degrees  higher  than  spore- 
free  cultures  (Foulerton).  It  is  probable  that  some  of  the 
spherical  bodies  formed  within  filaments  when  growing  in  the 
tissues  have  the  same  significance,  i.e.  are  gonidia,  whilst  others 
may  be  merely  the  result  of  degenerative  change.  Both  the 


CHARACTERS   OF   THE   ACTINOMYCES        289 

filaments   and    the    spherical    bodies    are    readily   stained    by 
Gram's  method. 

3.  Clubs. — These  are  elongated  pear-shaped  bodies  which  are 
seen  at  the  periphery  of  the  colony,  and  are  formed  by  a  sort  of 
hyaline  swelling  of  the  sheath  around  the  free  extremity  of  a 
filament  (Figs.  97,  98).  They  are  usually  homogeneous  and 
structureless  in  appearance.  In  the  human  subject  the  clubs  are 


Fia.  97. — Actinomyces  in  human  kidney,  showing  clubs  radially  arranged 

and  surrounded  by  pus.     The  filaments  had  practically  disappeared. 

Paraffin  section  ;  stained  with  hgerhatoxylin  and  rubin.      x  500. 

often  comparatively  fragile  structures,  which  are  easily  broken 
down,  and  may  sometimes  be  dissolved  in  water.  Sometimes 
they  are  well  seen  when  examined  in  the  fresh  condition,  but  in 
hardened  specimens  are  no  longer  distinguishable.  In  specimens 
stained  by  Gram's  method  they  are  usually  not  coloured  by  the 
violet,  but  take  readily  a  contrast  stain,  such  as  picric  acid, 
rubin,  etc;  sometimes  a  darkly-stained  filament  can  be  seen 
running  for  a  distance  in  the  centre,  and  may  have  a  knob-like 
extremity.  In  many  of  the  colonies  in  the  human  subject  the 
clubs  are  absent.  In  the  ox,  on  the  other  hand,  where  there  are 
19 


290     ACTINOMYCOSIS  AND   ALLIED   DISEASES 

much  older  colonies,  the  clubs  constitute  the  most  prominent 
feature,  whilst  in  most  colonies  the  filaments  are  more  or  less 
degenerated,  and  it  may  sometimes  be  impossible  to  find  any. 
They  often  form  a  dense  fringe  around  the  colony,  and  when 
stained  by  Gram's  method  retain  the  violet  stain.  They  have, 
in  fact,  undergone  some  further  chemical  change  which  produces 
the  altered  staining  reaction.  Occasionally  in  very  chronic 


FIG.  98.  — Colonies  of  actiuomyces,  showing  general  structural  arrangement 

and  clubs  at  periphery.     From  pus  in  human  subject. 

Stained  Gram  and  safranin.      x  60. 

lesions  in  the  human  subject  the  clubs  stain  with  Gram's 
method.  Clubs  showing  intermediate  staining  reaction  have 
been  described  in  the  ox  by  M'Fadyean.  The  club-formation 
probably  represents  a  means  of  defence  on  the  part  of  the 
parasite  against  the  phagocytes  of  the  tissue  :  the  view,  formerly 
held,  that  the  clubs  are  organs  of  fructification  has  now  been 
generally  abandoned. 

Tissue  Lesions. — In  the  human  subject  the  parasite  pro- 
duces by  its  growth  a  chronic  inflammatory  change,  usually 
ending  in  a  suppuration  which  slowly  spreads.  In  some  cases 


TISSUE   LESIONS  291 

there  is  a  comparatively  large  production  of  granulation  tissue, 
with  only  a  little  softening  in  the  centre,  so  that  the  mass  feels 
solid.  This  condition  is  sometimes  found  in  the  subcutaneous 
tissue,  especially  when  the  disease  has  not  advanced  far,  and 
also  in  dense  fibrous  tissue.  In  most  cases,  however,  and 
especially  in  internal  organs,  suppuration  is  the  outstanding 
feature ;  this  is  associated  with  abundant  growth  of  the  parasite 
in  the  filamentous  form  presenting  a  honeycomb  appearance. 
In  an  organ  such  as  the  liver,  multiple  foci  of  suppuration  are 
seen  at  the  spreading  margin  of  the  disease,  whilst  the  colonies 
of  the  parasite  may  be  seen  in  the  pus  with  the  naked  eye.  In 
the  older  parts  the  abscesses  have  become  confluent,  and  formed 
large  areas  of  suppuration.  The  pus  is  usually  of  greenish- 
yellow  colour,  and  of  somewhat  slimy  character. 

In  cattle  the  tissue  reaction  is  more  of  a  formative  type, 
there  being  abundant  growth  of  granulation  tissue,  which  may 
result  in  large  tumour-like  masses,  usually  of  more  or  less 
nodulated  character,  and  often  consisting  of  well-developed 
fibrous  tissue  containing  areas  of  younger  formation  in  which 
irregular  abscess  formation  is  usually  present.  The  cells  immedi- 
ately around  the  colonies  are  usually  irregularly  rounded,  or  may 
even  be  somewhat  columnar  in  shape,  whilst  farther  out  they 
become  spindle-shaped  and  concentrically  arranged.  It  is  not 
uncommon  to  find  leucocytes  or  granulation  tissue  invading  the 
substance  of  the  colonies,  and  portions  of  the  parasite,  etc.,  may 
be  contained  within  leucocytes  or  within  small  giant-cells  which 
are  sometimes  present.  A  similar  invasion  of  old  colonies  by 
leucocytes  is  sometimes  seen  in  human  actinomycosis. 

Origin  and  Distribution  of  Lesions. — The  lesions  in  the 
human  subject  may  occur  in  almost  any  part  of  the  body,  the 
paths  of  entrance  being  very  various.  In  many  cases  the 
entrance  takes  place  in  the  region  of  the  mouth  —  probably 
around  a  decayed  tooth — by  the  crypts  of  the  tonsil,  or  by  some 
abrasion.  Swelling  and  suppuration  may  then  follow  in  the 
vicinity  and  may  spread  in  various  directions.  The  periosteum 
of  the  jaw  or  the  vertebrae  may  thus  become  affected,  caries  or 
necrosis  resulting,  or  the  pus  may  spread  deeply  in  the  tissues  of 
the  neck,  and  may  even  pass  into  the  mediastinum.  Occasionally 
the  parasite  may  enter  the  tissues  from  the  oesophagus,  and  in  a 
considerable  number  of  cases  the  primary  lesion  is  in  some  part 
of  the  intestine,  generally  of  the  large  intestine.  The  parasite 
penetrates  the  wall  of  the  bowel,  and  may  be  found  deeply 
between  the  coats,  surrounded  by  purulent  material.  Thence  it 
may  spread  to  the  peritoneum  or  to  the  extraperitoneal  tissue, 


292      ACTINOMYCOSIS  AND   ALLIED   DISEASES 

the  retrocaecal  connective  tissue  and  that  around  the  rectum 
being  not  uncommonly  seats  of  suppuration  produced  in  this 
way.  A  peculiar  affection  of  the  intestine  has  been  described, 
in  which  slightly  raised  plaques  are  found  both  in  the  large  and 
small  intestines,  these  plaques  being  composed  almost  exclusively 
of  masses  of  the  actinomyces  along  with  epithelial  cells.  This, 
however,  is  a  rare  condition.  The  path  of  entrance  may  also  be 
by  the  respiratory  passages,  the  primary  lesion  being  pulmonary 
or  peribronchial ;  extensive  suppuration  in  the  lungs  may  result. 
Infection  may  also  occur  by  the  skin  surface,  and  lastly,  by  the 
female  genital  tract,  as  in  a  case  recorded  by  Grainger  Stewart 
and  Muir,  in  which  both  ovaries  and  both  Fallopian  tubes  were 
affected. 

When  the  parasite  has  invaded  the  tissues  by  any  of  these 
channels,  secondary  or  "  metastatic "  abscesses  may  occur  in 
internal  organs.  The  liver  is  the  organ  most  frequently  affected, 
though  abscesses  may  occur  in  the  lungs,  brain,  kidneys,  etc. 
In  such  cases  the  spread  takes  place  by  the  blood  stream,  and  it 
is  possible  that  leucocytes  may  be  the  carriers  of  the  infection, 
as  it  is  not  uncommon  to  find  leucocytes  in  the  neighbourhood 
of  a  colony  containing  small  portions  of  the  filaments  in  their 
interior. 

In  the  ox,  on  the  other  hand,  the  disease  usually  remains 
quite  local,  or  spreads  by  continuity.  It  may  produce  tumour- 
like  masses  in  the  region  of  the  jaw  or  neck,  or  it  may  specially 
affect  the  palate  or  tongue,  in  the  latter  producing  enlargement 
and  induration,  with  nodular  thickening  on  the  surface  —  the 
condition  known  as  "  woody  tongue." 

Source  of  the  Parasite. — There  is  a  considerable  amount  of 
evidence  to  show  that  outside  the  body  the  parasite  grows  on 
grain,  especially  on  barley.  Both  in  the  ox  and  in  the  pig  the 
parasite  has  been  found  growing  around  fragments  of  grain  em- 
bedded in  the  tissues.  There  are  besides,  in  the  case  of  the 
human  subject,  a  certain  number  of  cases  in  which  there  was  a 
history  of  penetration  of  a  mucous  surface  by  a  portion  of  grain, 
and  in  a  considerable  proportion  of  cases  the  patient  has  been 
exposed  to  infection  from  this  source.  The  position  of  the 
lesions  in  cattle  is  also  in  favour  of  such  a  view. 

Cultivation  (for  methods  of  isolation  see  later). — The  de- 
scriptions of  the  cultures  obtained  by  various  investigators  differ 
in  essential  particulars,  and  there  is  no  doubt  that  the  organisms 
described  are  different.  The  following  is  the  account  of  the 
organism  as  cultivated  by  Bostrom  : — 

On  agar  or  glycerin  agar  at   37°  C.,  growth  is  generally 


CULTIVATION   OF   ACTINOMYCES 


293 


visible  on  the  third  or  fourth  day  in  the  form  of  little  transparent 
drops  which  gradually  enlarge  and  form  rounded  projections  of 
a  reddish-yellow  tint  and  somewhat  transparent  appearance,  like 
drops  of  amber.  The  growths  tend  to  remain  separate,  and  even 
when  they  become  confluent,  the  nodular  character  is  maintained. 
They  have  a  tough  con- 
sistence, being  with  diffi- 
culty broken  up,  and  ad- 
here firmly  to  the  surface 
of  the  agar.  Older  growths 
often  show  on  the  surface 
a  sort  of  corrugated  aspect, 
and  may  sometimes  pre- 
sent the  appearance  of 
having  been  dusted  with 
a  brownish-yellow  powder 
(Fig.  99).  ' 

In  the  cultures  at  an 
early  stage  the  growth  is 
composed  of  branching 
filaments,  which  stain 
uniformly  (Fig.  100),  but 
later  some  of  the  super- 
ficial filaments  may  show 
segmentation  into  gonidia. 
Slight  bulbous  thicken- 
ings may  be  seen  at  the 
end  of  some  of  the  fila- 
ments, but  true  clubs 
have  not  been  observed.  A  B 

On  gelatin  the  same  FIG.  99. — Cultures  of  the  actinomyces  on 
tendency  to  grow  in  little  glycerin  agar,  of  about  three  weeks'  growth, 
SDherical  masses  is  seen  showing  the  appearances  which  occur.  The 

,  growth  in  A  is  at  places  somewhat  corru- 

and  the  medium  becomes         gated  on  the  sllrface.    Natural  size. 
very      slowly      liquefied. 

When  this  occurs  the  liquefied  portion  has  a  brownish  colour 
and  somewhat  syrupy  consistence,  and  the  growths  may  be  seen 
at  the  bottom,  as  little  balls,  from  the  surface  of  which  filaments 
radiate. 

The  organism  obtained  in  culture  by  Wolff  and  Israel  (vide 
infra)  is  probably  the  same  as  the  one  which  has  been  recently 
described  in  detail  by  J.  H.  Wright,  who  obtained  it  in  pure  con- 
dition from  fifteen  different  cases  of  the  disease.  It  differs  markedly 
from  Bostrom's  organism  in  being  almost  a  strict  anaerobe  and  in 


294     ACTINOMYCOSIS   AND   ALLIED   DISEASES 


ceasing  to  grow  at  a  temperature  a  little  below  that  of  the  body. 
Under  ordinary  aerobic  conditions  either  no  growth  occurs  or 
it  is  of  a  very  slight  character.  On  the  surface  of  agar  under 
anaerobic  conditions  the  organism  produces  dense  rounded 
colonies  of  greyish -white  colour,  which  sometimes  assume  a 
rosette  form.  A  somewhat  curious  feature  of  growth  is  described 
by  Wright,  namely,  that  in  a  shake  culture  in  glucose  agar  the 
colonies  are  most  numerous,  and  form  a  dense  zone  about  half 
an  inch  from  the  surface  of  the  medium,  that  is,  at  a  level 

where  there  is  presum- 
ably a  mere  trace  of 
oxygen  obtainable  (Fig. 
1 0 1 ).  In  bouillon,  growth 
takes  place  at  the  bottom 
of  the  medium  in  rounded 
masses  which  afterwards 
undergo  disintegration. 
Wright  found  when  the 
organism  was  grown  in 
the  presence  of  serum  or 
other  animal  fluids  that 
the  formation  of  true 
clubs  occurred  at  the  ex- 
tremity of  some  of  the  fila- 
ments (Fig.  102).  From 
the  conditions  under 
FIQ.  100.— Actinomyces,  from  a  culture  on  which  growth  occurs,  he 

fifmlmeTs?  Sll°Wing  the  branCWng  °f  *     Alined     to     regard 
Stained  with  fuchsin.     x  1000.  it    as    a    true    parasite, 

and    doubts   whether   it 

can  have  a  saprophytic  existence  outside  the  body,  e.g.  on  grain. 
He  is  also  of  opinion  that  all  cases  of  true  actinomycosis,  i.e.  cases 
where  colonies  visible  to  the  naked  eye  are  present,  are  probably 
produced  by  one  species,  and  that  the  aerobic  organisms  obtained 
by  Bostrom  and  others  are  probably  accidental  contaminations. 
It  is  quite  evident  that  further  investigations  are  required  in 
the  light  of  the  results  detailed.  Certainly  the  parasite  in 
many  cases  of  actinomycosis  in  the  human  subject  does  not 
grow  on  ordinary  media  under  aerobic  conditions  as  Bostrom's 
organism  does. 

Varieties  of  Actinomyces  and  Allied  Forms. — It  is  probable  that  in 
the  cases  of  the  disease  described  in  the  human  subject  there  is  more 
than  one  variety  or  species  of  parasite  belonging  to  the  same  group. 
Gasperini  has  described  several  varieties  of  actinomyces  bows  according 


VARIETIES   OF  ACTINOMYCES 


295 


to  the  colour  of  the  growths, 
and  a  similar  condition  may 
obtain  in  the  case  of  the 
human  subject.  Further- 
more a  considerable  number 
of  streptothrices  have  been 
found  in  cases  of  disease  in 
the  human  subject,  the  as- 
sociated lesions  varying  in 
character  from  tubercle-like 
nodules  on  the  one  hand  to 
suppurative  processes  on  the 
other.  The  organisms  culti- 
vated from  such  sources 
differ  according  to  their 
microscopic  characters  (for 
example,  some  form  "clubs" 
whilst  others  do  not)  ac- 
cording to  their  conditions 
of  growth,  staining  reac- 
tions, etc.  Of  these  only  a 
few  examples  may  here  be 
mentioned,  but  it  may  be 
noted  that  the  importance 
of  the  streptothrices  as 
causes  of  disease  is  con- 
stantly being  extended. 
Wolff  and  Israel  cultivated 
from  two  cases  of  "actino- 
mycosis "  in  man  a  strep- 
tothrix  which  differs  in  so 
many  important  points  from 
the  actinomyces  of  Bostrom 


FIG.  102. — Section  of  a  colony  of  actinomyces 
from  a  culture  in  blood  serum,  showing  the 
formation  of  clubs  at  the  periphery.  x  1500. 


Fia.  101.1 — Shake  cultures  of  actinomyces  in 
glucose  agar,  showing  the  maximum 
growth  at  some  distance  from  the  sur- 
face of  the  medium. 


that  it  is  now  regarded 
as  a  distinct  species.  An- 
other species  was  culti- 
vated by  Eppinger  from  a 
brain  abscess,  and  called 
by  him  "cladothrixaster- 
oides,"  from  the  appear- 
ance of  its  colonies  on 
culture  media.  A  case 
of  general  streptothrix 
infection  in  the  human 
subject  described  by 
MacDonald  was  probably 
due  to  the  same  organism 
as  Eppinger's.  In  the 
tissues  it  grows  in  a 
somewhat  diffuse  manner 
and  does  not  form  clubs ; 


1  For  Figs.  101  and  102 
we  are  indebted  to  Dr.  J.  Homer  Wright  of  Boston,  U.S.A. 


296     ACTINOMYCOSIS  AND  ALLIED  DISEASES 

in  rabbits  and  guinea-pigs  it  produces  tubercle-like  lesions.  Flexner 
observed  a  streptothrix  in  the  lungs  associated  with  lesions  somewhat  like 
a  rapid  phthisis,  and  applied  the  name  ' '  pseudo-tuberculosis  hominis 
streptothricea  "  ;  an  apparently  similar  condition  has  been  described  by 
Buchholz.  Berestnew  cultivated  two  species  of  streptothrix  from 
suppurative  lesions,  one  of  which  is  acid -fast  and  grows  only  in 
anaerobic  conditions.  Birt  and  Leishman  have  described  another  acid- 
fast  streptothrix  obtained  from  cirrhotic  nodules  in  the  lungs  of  a  man. 
This  organism  grows  readily  on  ordinary  media,  forming  a  white 
powdery  growth  which  afterwards  assumes  a  pinkish  colour  ;  it  is 
pathogenic  for  guinea-pigs,  in  which  it  causes  caseous  lesions.  There 
is,  further,  the  streptothrix  maduree  described  below. 

In  diseases  of  the  lower  animals  several  other  forms  have  been  found. 
For  example,  a  streptothrix  has  been  shown  by  Nocard  to  be  the  cause 
of  a  disease  of  the  ox, — "farcin  du  bceuf," — a  disease  in  which  also 
there  occur  tumour -like  masses  of  granulation  tissue.  Dean  has 
cultivated  from  a  nodule  in  a  horse  another  streptothrix,  which  produces 
tubercle-like  nodules  in  the  rabbit  with  club-formation  ;  it  has  close 
resemblances  to  the  organism  of  Israel  and  Wolff.  The  so-called 
diphtheria  of  calves  and  "bacillary  necrosis"  in  the  ox  are  probably 
both  produced  by  another  streptothrix  or  leptothrix,  which  grows 
diffusely  in  the  tissues  in  the  form  of  fine  felted  filaments.  Further 
investigation  may  show  that  some  of  these  or  other  species  may  occur 
in  the  human  subject  in  conditions  which  are  not  yet  differentiated. 

Experimental  Inoculation. — Inoculation  of  smaller  animals, 
such  as  rabbits  and  guinea-pigs,  has  usually  failed  to  give  positive 
results.  This  was  the  case,  for  example,  in  the  important  series 
of  experiments  by  Bostrom,  and  it  may  be  assumed  that  these 
animals  are  little  susceptible  to  the  actinomyces.  The  disease 
has,  however,  been  experimentally  produced  in  the  bovine  species 
both  by  cultures  from  the  ox  and  from  the  human  subject. 
Inoculation  with  the'  organism  of  Israel  and  Wolff  produces 
nodular  lesions  both  in  rabbits  and  in  guinea-pigs,  while  Wright 
found  that  characteristic  colonies  and  lesions  resulted  although 
the  parasite  did  not  grow  to  any  great  extent.  Several  of  the 
other  species  of  streptothrix  have  been  found  to  possess  active 
pathogenic  properties. 

Methods  of  Examination  and  Diagnosis. — As  actinomycosis 
cannot  be  diagnosed  with  certainty  apart  from  the  discovery  of 
the  parasite,  a  careful  examination  of  the  pus  in  obscure  cases 
of  suppuration  should  always  be  undertaken.  As  already  stated, 
the  colonies  can  be  recognised  with  the  naked  eye,  especially 
when  some  of  the  pus  is  spread  out  on  a  piece  of  glass.  If  one 
of  these  is  washed  in  salt  solution  and  examined  unstained,  the 
clubs,  if  present,  are  at  once  seen  on  microscopic  examination. 
Or  the  colony  may  be  stained  with  a  simple  reagent  such  as 
picrocarmine,  and  mounted  in  glycerin  or  Farrant's  solution. 
To  study  the  filaments,  a  colony  should  be  broken  down  on  a 


MADURA   DISEASE  297 

cover-glass,  dried,  and  stained  with  a  simple  solution  of  any  of 
the  basic  aniline  dyes,  such  as  gentian-violet,  though  better 
results  are  obtained  by  carbol-thionin-blue,  or  by  carbol-fuchsin 
diluted  with  five  parts  of  water.  If  the  specimen  be  over-stained, 
it  may  be  decolorised  by  weak  acetic  acid.  Cover-glass  prepara- 
tions of  this  kind,  and  also  of  cultures,  are  readily  stained  by 
these  methods,  but  in  the  case  of  sections  of  the  tissues  Gram's 
method,  or  a  modification  of  it,  should  be  used  to  show  the 
filaments,  etc.,  a  watery  solution  of  acid  fuchsin  being  afterwards 
used  to  stain  the  clubs.  By  this  method,  very  striking  prepara- 
tions may  be  obtained. 

Cultures  should  be  made  both  under  aerobic  and  anaerobic 
conditions.  Tubes  of  agar  or  glycerin  agar  should  be  inoculated 
and  incubated  at  37°  C.  ;  deep  tubes  of  melted  glucose  agar 
should  also  be  used,  the  inoculated  material  being  diffused 
through  the  medium,  separate  colonies  may  thus  be  obtained. 
Owing  to  the  slow  growth  of  the  actinomyces,  however,  the 
obtaining  of  pure  cultures  is  difficult,  unless  the  pus  is  free  from 
contamination  with  other  organisms. 


MADTJKA  DISEASE. 

Madura  disease  or  mycetoma  resembles  actinomycosis  both 
as  regards  the  general  characters  of  the  lesions  and  the  occurrence 
of  the  parasite  in  the  form  of  colonies  or  "  granules."  There  is 
no  doubt,  however,  that  the  two  conditions  are  distinct,  and  it 
also  appears  established  that  the  two  varieties  of  Madura  disease 
(vide  infra)  are  produced  by  different  organisms.  This  disease 
is  comparatively  common  in  India  and  in  various  other  parts  of 
the  tropics  :  it  has  also  been  met  with  in  Algiers  and  in  America. 
Madura  disease  differs  from  actinomyces  not  only  in  its  geo- 
graphical distribution  but  also  in  its  clinical  characters.  Its 
course,  for  example,  is  of  an  extremely  chronic  nature,  and 
though  the  local  disease  is  incurable  except  by  operation,  the 
parasite  never  produces  secondary  lesions  in  internal  organs. 
Vincent  also  found  that  iodide  of  potassium,  which  has  a  high 
value  as  a  therapeutic  agent  in  many  cases  of  actinomycosis,  had 
no  effect  in  the  case  of  Madura  disease  studied  by  him.  It  most 
frequently  affects  the  foot ;  hence  the  disease  is  often  spoken  of 
as  "Madura  foot."  The  hand  is  rarely  affected.  In  the  parts 
affected  there  is  a  slow  growth  of  granulation  tissue  which  has  an 
irregularly  nodular  character,  and  in  the  centre  of  the  nodules 
there  occurs  purulent  softening  which  is  often  followed  by  the 


298    ACTINOMYCOSIS  AND   ALLIED   DISEASES 


formation  of  fistulous  openings  and  ulcers.  There  are  great  en- 
largement and  distortion  of  the  part  and  frequently  caries  and 
necrosis  of  the  bones.  Within  the  softened  cavities  and  also  in 
the  spaces  between  the  fibrous  tissue,  small  rounded  bodies  or 
granules,  bearing  a  certain  resemblance  to  the  actinomyces,  are 
present.  These  may  have  a  yellowish  or  pinkish  colour,  com- 
pared from  their  appearance  to  fish  roe,  or  they  may  be  black 
like  grains  of  gunpowder,  and  may  by  their  conglomeration 
form  nodules  of  considerable  size.  Hence  a  pale  variety  and 
a  black  variety  of  the  disease  have  been  distinguished ;  in  both 
varieties  the  granules  mentioned  reach  a  rather  larger  size 

than  in  actinomycosis. 
These  two  conditions  will 
be  considered  separately. 
Pale  Variety. — When 
the  roe-like  granules  are 
examined  microscopic- 
ally, they  are  found,  like 
the  actinomyces,  to  show 
in  their  interior  an  abun- 
dant mass  of  branching 
filaments  with  mycelial 
arrangement.  There  may 
also  be  present  at  the 
periphery  club-like  struc- 
tures, as  in  actinomyces ; 
sometimes  they  are  ab- 
sent. These  structures 
often  have  an  elongated 

agar.      Stained  with  carbol-thionin-blue.    wedge-shape,  iorming  an 
x  1000.  outer  zone  to  the  colony, 

and  in   some   cases   the 

filaments  can  be  found  to  be  connected  with  them.  Vincent 
obtained  cultures  of  the  parasite  from  a  case  in  Algiers,  and  found 
it  to  be  a  distinct  species :  it  is  now  known  as  the  streptothrix 
Madurce.  Morphologically  it  closely  resembles  the  actinomyces, 
but  it  presents  certain  differences  in  cultural  characters.  In 
gelatin  it  forms  raised  colonies  of  a  yellowish  colour,  with 
umbilication  of  the  centre,  and  there  is  no  liquefaction  of  the 
medium.  On  agar  the  growth  assumes  a  reddish  colour ;  the 
organism  flourishes  well  in  various  vegetable  infusions  in  which 
the  actinomyces  does  not  grow.  On  all  the  media  growth  only 
takes  place  in  aerobic  conditions.  Experimental  inoculation  of 
various  animals  has  failed  to  reproduce  the  disease.  There  is 


MADURA  DISEASE  299 

therefore    no    doubt   that    the   streptothrix   madurae   and    the 
actinomyces  are  distinct  species. 

Black  Variety. — The  observations  of  J.  H.  Wright,  who 
obtained  pure  cultures  of  a  hyphomycete,  show  that  this  variety  is 
a  distinct  affection  from  the  pale  variety.  The  pigment  may  be 
dissolved  by  soaking  the  granules  for  a  few  minutes  in  hypochlorite 
of  sodium  solution,  and  the  granules  may  then  be  crushed  out 
beneath  a  cover-glass  and  examined  microscopically.  The  black 
granules  are  composed  of  a  somewhat  homogeneous  ground- 
substance  impregnated  with  pigment,  and  in  this  there  is  a 
mycelium  of  thick  filaments  or  hyphse,  many  of  the  segments  of 
which  are  swollen;  at  the  periphery  the  hyphse  form  a  zone 
with  radiate  arrangement.  In  many  of  the  older  granules  the 
parasite  is  largely  degenerated  and  presents  an  amorphous  appear- 
ance. Wright  planted  over  sixty  of  the  black  granules  in  various 
culture  media,  and  obtained  cultures  of  a  hyphomycete  from 
about  a  third  of  these.  The  organism  grows  well  on  agar, 
bouillon,  potato,  etc.  ;  on  agar  it  forms  a  felted  mass  of  greyish 
colour,  and  in  old  cultures  black  granules  appear  amongst  the 
mycelium.  Microscopically  the  parasite  appears  as  a  mycelium 
of  thick  branching  filaments  with  delicate  transverse  septa ;  in 
the  older  threads  the  segments  become  swollen,  so  that  strings 
of  oval-shaped  bodies  result.  No  signs  of  spore-formation  were 
noted.  Inoculation  of  animals  with  cultures  gave  negative 
results,  as  did  also  direct  inoculation  with  the  black  granules 
from  the  tissues. 


CHAPTER  XIII 

ANTHRAX.1 

OTHER     NAMES. — SPLENIC     FEVER,     MALIGNANT     PUSTULE,     WOOL- 

SORTER'S  DISEASE.  GERMAN,  MILZBRAND;  FRENCH,  CHARBON.2 

Introductory. — Anthrax  is  a  disease  occurring  epidemically 
among  the  herbivora,  especially  sheep  and  oxen,  in  which 
animals  it  has  the  characters  of  a  rapidly  fatal  form  of 
septicaemia  with  splenic  enlargement,  attended  by  an  extensive 
multiplication  of  characteristic  bacilli  throughout  the  blood. 
The  disease  does  not  occur  as  a  natural  affection  in  man,  but  may 
be  communicated  to  him  directly  or  indirectly  from  animals,  and 
it,  may  then  appear  in  certainly  two  and  possibly  three  forms.  In 
the  first  there  is  infection  through  the  skin,  in  which  a  local  lesion, 
the  "  malignant  pustule,"  occurs.  In  the  second  form  infection 
takes  place  through  the  respiratory  tract.  Here  very  aggravated 
symptoms  centred  in  the  thorax,  with  rapidly  fatal  termination, 
follow.  Thirdly,  an  infection  may  probably  take  place  through 
the  intestinal  tract,  which  is  now  the  first  part  to  give  rise  to 
symptoms.  In  all  these  forms  of  the  affection  in  the  human 
subject,  the  bacilli  are  in  their  distribution  much  more  re- 
stricted to  the  local  lesions  than  is  the  case  in  the  ox,  their 
growth  and  spread  being  attended  by  inflammatory  oedema 
and  often  by  haemorrhages. 

Historical  Summary. — Historical  researches  leave  little  doubt  that 
from  the  earliest  times  anthrax  has  occurred  among  cattle.  For  a  long 
time  its  pathology  was  not  understood,  and  it  went  by  many  names. 

1  In  even  recent  works  on  surgery   the  term  "anthrax"  may  be  found 
applied  to  any  form  of  carbuncle.     Before  its  true  pathology  was  known  the 
local   variety   of  the  disease  which  occtirs  in  man  and  which  is  now  called 
"malignant  pustule"  was  known  as  "malignant  carbuncle." 

2  This  must  be  distinguished  from  "charbon  symptomatique, "  which  is 
quite  a  different  disease. 

300 


BACILLUS   ANTHRACIS  301 

During  the  early  part  of  last  century  much  attention  was  paid  to 
it,  and,  with  a  view  to  finding  out  its  nature  and  means  of  spread, 
various  conditions  attaching  to  its  occurrence,  such  as  those  of  soil  and 
weather,  were  exhaustively  studied.  Pollender  in  1849  pointed  out  that 
the  blood  of  anthrax  animals  contained  numerous  rod-shaped  bodies 
which  he  conjectured  had  some  causal  connection  with  the  disease.  In  1863 
Davaine  announced  that  they  were  bacteria,  and  originated  the  name 
bacillus  anthracis.  He  stated  that  unless  blood  used  in  inoculation 
experiments  on  animals  contained  them,  death  did  not  ensue.  Though 
this  conclusion  was  disputed,  still  by  the  work  of  Davaine  and  others 
the  causal  relationship  of  the  bacilli  to  the  disease  had  been  nearly 
established  when  the  work  of  Koch  appeared  in  1876.  This  constituted 
that  observer's  first  contribution  to  bacteriology,  and  did  much  to  clear  up 
the  whole  subject.  Koch  confirmed  Davaine's  view  that  the  bodies  were 
bacteria.  He  observed  in  the  blood  of  anthrax  animals  the  appearance 
of  division,  and  from  this  deduced  that  multiplication  took  place  in  the 
tissues.  He  observed  them  under  the  microscope  dividing  outside  the 
body,  and  noticed  spore  formation  taking  place.  He  also  isolated  the 
bacilli  in  pure  culture  outside  the  body,  and,  by  inoculating  animals  with 
them,  produced  the  disease  artificially.  In  his  earlier  experiments  he 
failed  to  produce  death  by  feeding  susceptible  animals  both  with  bacilli 
and  spores,  and  as  the  intestinal  tract  was,  in  his  view,  the  natural  path 
of  infection,  he  considered  as  incomplete  the  proof  of  this  method  of  the 
spontaneous  occurrence  of  anthrax  in  herds  of  animals.  Koch's  obsterva- 
tions  were,  shortly  afterwards,  confirmed  in  the  main  by  Pasteur, 
though  controversy  arose  between  them  on  certain  minor  points. 
Moreover,  further  research  showed  that  the  disease  could  be  produced 
in  animals  by  feeding  them  with  spores,  and  thus  the  way  in  which  the 
disease  might  spread  naturally  was  explained. 

The  Bacillus  Anthracis. — Anthrax  as  a  disease  in  man  is  of 
comparative  rarity.  Not  only,  however,  is  the  bacillus  anthracis 
easy  of  growth  and  recognition,  but  in  its  growth  it  illustrates 
many  of  the  general  morphological  characters  of  the  whole  group 
of  bacilli,  and  is  therefore  of  the  greatest  use  to  the  student. 
Further,  its  behaviour  when  inoculated  in  animals  illustrates 
many  of  the  points  raised  in  connection  with  such  difficult 
questions  as  the  general  pathogenic  effects  of  bacteria,  immunity, 
etc.  Hence  an  enormous  amount  of  work  has  been  done  in 
investigating  it  in  all  its  aspects. 

If  a  drop  of  blood  is  taken  immediately  after  death  from  an 
auricular  vein  of  a  cow,  for  example,  which  has  died  from 
anthrax,  and  examined  microscopically,  it  will  be  found  to 
contain  a  great  number  of  large  non-motile  bacilli.  On  making 
a  cover-glass  preparation  from  the  same  source,  and  staining  with 
watery  methylene-blue,  the  characters  of  the  bacilli  can  be  better 
made  out.  They  are  about  1*2  /x  thick  or  a  little  thicker,  and  6 
to  8  \i  long,  though  both  shorter  and  longer  forms  also  occur. 
The  ends  are  sharply  cut  across,  or  may  be  slightly  dimpled  so 
as  to  resemble  somewhat  the  proximal  end  of  a  phalanx.  Their 


302 


ANTHRAX 


protoplasm  is  very  finely  granular,  and  sometimes  appears  sur- 
rounded by  a  thin  unstained  capsule.  When  several  bacilli  lie 
end  to  end  in  a  thread,  the  capsule  seems  common  to  the  whole 
thread  (Fig.  108).  They  stain  well  with  all  the  basic  aniline  dyes 
and  are  not  decolorised  by  Gram's  method. 

Plate  Cultures. — From  a  source  such  as  that  indicated,  it  is 
easy  to  isolate  the  bacilli  by  making  gelatin  or  agar  plates.  If, 
after  twelve  hours'  incubation  at  37°  C.,  the  latter  be  examined 
under  a  low  objective,  colonies  will  be  observed.  They  are  to 
be  recognised  by  beautiful  wavy  wreaths  like  locks  of  hair, 
radiating  from  the  centre  and  apparently  terminating  in  a  point 

which,  however,  on  ex- 
amination with  a  higher 
power  is  observed  to  be  a 
filament  which  turns  upon 
itself  (Fig.  1 04).  The  whole 
colony  is,  in  fact,  probably 
one  long  thread.  Such 
colonies  are  very  suitable 
for  making  impression  pre- 
parations (vide  p.  118) 
which  preserve  perman- 
ently the  appearances  de- 
scribed. On  examining 
such  with  a  high  power, 
the  wreaths  are  seen  to  be 
made  up  of  bundles  of 
FIG.  104.— Surface  colony  of  the  anthrax  l°ng  filaments  lying  par- 
bacillus  on  an  agar  plate,  showing  the  allel  with  one  another,  each 
characteristic  appearances,  x  30.  filament  consisting  of  a 

chain  of  bacilli  lying  end 
to  end,  and  similar  to  those  observed  in  the  blood  (Fig.  105). 

On  gelatin  plates,  after  from  twenty-four  to  thirty-six  hours 
at  20°  C.,  the  same  appearances  manifest  themselves,  and  later 
they  are  accompanied  by  liquefaction  of  the  gelatin.  In  gelatin 
plates,  however,  instead  of  the  characteristically  wreathed  appear- 
ance at  the  margin,  the  colonies  sometimes  give  off  radiating 
spikelets  irregularly  nodulated,  which  produce  a  star-like  form. 
These  spikelets  are  composed  of  spirally  twisted  threads. 

From  such  plates  the  bacilli  can  be  easily  isolated,  and  the 
appearances  of  pure  cultures  on  various  media  studied. 

In  bouillon,  after  twenty-four  hours'  incubation  at  37°  C., 
there  is  usually  the  appearance  of  irregularly  spiral  threads 
suspended  in  the  liquid.  These,  on  being  examined,  are  seen 


BACILLUS  ANTHRACIS 


303 


to  be  made  up  of  bundles  of  parallel  chains  of  bacilli.  Later, 
growth  is  more  abundant,  and  forms  a  flocculent  mass  at  the 
bottom  of  the  fluid. 

In  gelatin  stab  cultures,  the  characteristic  appearance  can  be 
best  observed  when  a  low  proportion,  say  7J  per  cent,  of  gelatin 


FIG.  105. — Anthrax  bacilli,  arranged  in  chains, 
from  a  twenty-four  hours'  culture  on  agar 
at  37°  C. 

Stained  with  fuchsin.      x  1000. 

is  present,  and  when  the  tube  is  directly 

inoculated    from    anthrax    blood.       In 

about  two  days  there   radiate  out  into 

the    medium     from    the    needle    track 

numberless    very    fine    spikelets    which 

enable    the  cultures  to  be  easily  recog-    FlG.i06.-Stab  culture  of 

nised.      These   spikelets  are  longest  at 

the   upper    part    of    the   needle    track 

(Fig.    106).     Not    much    spread    takes 

place    on    the    surface    of    the  gelatin, 

but  here   liquefaction   commences,    and 

gradually    spreads    down  the  stab   and 

out   into   the   medium,    till   the   whole 

of  the  gelatin  may  be  liquefied.     Gelatin  slope  cultures  exhibit 

a  thick  felted  growth,  the  edges  of  which   show   the   wreathed 

appearance   seen    in   plate   cultures.      Liquefaction   here  soon 

ploughs  a  trough  in  the  surface  of  the  medium.     Sometimes 

"spiking"  does  not  take  place  in  gelatin   stab   cultures,  only 


the  anthrax  bacillus  in 
peptone-gelatin  ;  seven 
days'  growth.  It  shows 
the  "spiking"  and  also, 
at  the  surface,  com- 
mencing liquefaction. 
Natural  size. 


304  ANTHRAX 

little  round  particles  of  growth  occurring  down  the  needle  track, 
followed  by  liquefaction.  As  has  been  shown  by  Richard  Muir, 
this  property  of  spiking  can  be  restored  by  growing  the  bacillus 
for  twenty-four  hours  on  blood  agar  at  37°  C.  Agar  sloped 
cultures  have  the  appearance  of  similar  cultures  in  gelatin, 
though,  of  course,  no  liquefaction  takes  place. 

Blood  serum  sloped  cultures  present  the  same  appearances  as 
those  on  agar.  The  margin  of  the  surface  growth  on  any  of  the 
solid  media  shows  the  characteristic  wreathing  seen  in  plate 
colonies. 

On  potatoes  there  occurs  a  thick  felted  white  mass  of  bacilli 
showing  no  special  characters.  Such  a  growth,  however,  is  useful 
for  studying  sporulation. 

The  anthrax  bacillus  will  thus  grow  readily  on  any  of  the 
ordinary  media.  It  can  usually  be  sufficiently  recognised  by  its 
microscopic  appearance,  by  its  growth  on  agar  or  gelatin  plates, 
and  by  its  growth  in  gelatin  stab  cultures.  The  growth  on 
plates  is  specially  characteristic,  and  is  simulated  by  no  other 
pathogenic  organism. 

The  Biology  of  the  B.  Anthracis. — Koch  found  that  the 
bacillus  anthracis  grows  best  at  a  temperature  of  35°  C.  Growth, 
i.e.  multiplication,  does  not  take  place  below  12°  C.  or  above 
45°  C.  In  the  spore-free  condition  the  bacilli  have  comparatively 
low  powers  of  resistance.  They  do  not  stand  long  exposure  to 
60°  C.,  and  if  kept  at  ordinary  temperature  in  the  dry  condition 
they  are  usually  found  to  be  dead  after  a  few  days.  The  action 
of  the  gastric  juice  is  rapidly  fatal  to  them,  and  they  are  accord- 
ingly destroyed  in  the  stomachs  of  healthy  animals.  They  are 
also  soon  killed  in  the  process  of  putrefaction.  They  can,  how- 
ever, be  cooled  below  the  freezing-point  without  dying.  The 
bacillus  can  grow  without  oxygen,  but  some  of  its  vital  functions 
are  best  carried  on  in  the  presence  of  this  gas.  Thus  in  anthrax 
cultures  the  liquefaction  of  gelatin  always  commences  at  the 
surface  and  spreads  downwards.  Growth  is  more  rapid  in  the 
presence  of  oxygen,  and  spore  formation  does  not  occur  in  its 
absence.  The  organism  may  be  classed  as  a  facultative  anaerobe. 

Sporulation. — Under  certain  circumstances  sporulation  occurs 
in  anthrax  bacilli.  The  morphological  appearances  are  of  the 
ordinary  kind.  A  little  highly  refractile  speck  appears  in  the 
protoplasm  about  the  centre  of  the  bacillus;  this  gradually 
increases  in  size  until  it  forms  an  oval  body  about  the  same 
thickness  as  the  bacillus  lying  in  the  bacillary  protoplasm  (Fig. 
107).  The  Blatter  gradually  loses  its  staining  capacities  and 
finally  disappears.  The  spore  thus  lies  free  as  an  oval  highly 


BIOLOGY   OF   THE   B.   ANTHRACIS 


305 


refractile  body  which  does  not  stain  by  ordinary  methods,  but 
which  can  be  easily  stained  by  the  special  methods  described 
for  such  a  purpose  (p.  102).  When  the  spore  is  again  about  to 
assume  the  bacillary  form  the  capsule  is  apparently  absorbed, 
and  the  protoplasm  within  grows  out,  taking  on  the  ordinary  rod- 
shaped  form. 

According  to  most  observers  sporulation  never  occurs  within 
the  body  of  an  animal  suffering  from  anthrax.  Koch  attributes 
this,  probably  rightly,  to  the  absence  of  free  oxygen.  The  latter 
gas  he  found  necessary  to  the  occurrence  of  spores  in  cultures 
outside  the  body.  Many,  however,  are  inclined  to  assign  as  the 
cause  of  sporulation  the 
absence  of  the  optimum 
pabulum,  which  in  the  case 
of  anthrax  is  afforded  by 
the  animal  tissues.  Besides 
these  conditions  there  is 
another  factor  necessary 
to  sporulation,  viz.  a  suit- 
able temperature.  The 
optimum  temperature  for 
spore  production  is  30°  C. 
Koch  found  that  spore 
formation  did  not  occur 
below  18°  C.  Above  42° 
C.  not  only  does  sporula- 
tion cease,  but  Pasteur 
found  that  if  bacilli  were 
kept  at  this  temperature 
for  eight  days  they  did  not  gtained  with 
regain  the  capacity  when  blue,  x  1000. 
again  grown  at  a  lower 

temperature.  In  order  to  make  them  again  capable  of  sporing 
it  is  necessary  to  adopt  special  measures,  such  as  passage  through 
the  bodies  of  a  series  of  susceptible  animals. 

Anthrax  spores  have  extremely  high  powers  of  resistance. 
In  a  dry  condition  they  will  remain  viable  for  a  year  or  more. 
Koch  found  they  resisted  boiling  for  five  minutes  ;  and  dry  heat 
at  140°  C.  must  be  applied  for  several  hours  to  kill  them  with 
certainty.  Unlike  the  bacilli,  they  can  resist  the  action  of  the 
gastric  juice  for  a  long  period  of  time.  They  are  often  used 
as  test  objects  by  which  the  action  of  germicides  is  judged.  For 
this  purpose  an  emulsion  is  made  by  scraping  off  a  surface 
culture  and  rubbing  it  up  in  a  little  sterile  water.  Into  this 
20 


FIG.  107. — Anthrax  bacilli  containing  spores 
(the  darkly  coloured  bodies)  ;  from  a 
three  days'  culture  on  agar  at  37°  C. 

and  methylene- 


306  ANTHRAX 

sterile  silk  threads  are  dipped,  which,  after  being  dried  over 
strong  sulphuric  acid  in  a  desiccator,  can  be  kept  for  long 
periods  of  time  in  an  unchanged  condition.  For  use  they  are 
placed  in  the  germicidal  solution  for  the  desired  time,  then 
washed  with  water  to  remove  the  last  traces  of  the  reagent  and 
laid  on  the  surface  of  agar  or  placed  in  bouillon,  in  order  that  if 
death  has  not  occurred  growth  may  be  observed  (see  Chap.  IV.). 

Anthrax  in  Animals. — Anthrax  occurs  from  time  to  time 
epidemically  in  sheep,  cattle,  and,  more  rarely,  in  horses  and 
deer.  These  epidemics  are  found  in  various  parts  of  the  world, 
although  they  are  naturally  most  far-reaching  where  legal  pre- 
cautions to  prevent  the  spread  of  infection  are  non-existent. 
All  the  countries  of  Europe  are  from  time  to  time  visited  by  the 
disease,  but  in  some  it  is  much  more  common  than  in  others. 
In  Britain  the  death-rate  is  small,  but  in  France  the  annual 
mortality  among  sheep  was  probably  10  per  cent  of  the  total 
number  in  the  country,  and  among  cattle  5  per  cent.  These 
figures,  however,  have  been  largely  modified  by  the  system  of 
preventive  treatment  which  will  be  presently  described.  In  sheep 
and  cattle  the  disease  is  specially  virulent.  An  animal  may 
suddenly  drop  down,  with  symptoms  of  collapse,  quickening  of 
pulse  and  respiration,  and  dyspnoea,  and  death  may  occur  in 
a  few  minutes.  In  less  acute  cases  the  animal  is  apparently  out 
of  sorts,  and  does  not  feed ;  its  pulse  and  respiration  are 
quickened ;  rigors  occur,  succeeded  by  high  temperature ;  there 
is  a  sanguineous  discharge  from  the  bowels,  and  bloody  mucus 
may  be  observed  about  the  inouth  and  nose.  There  may  be 
convulsive  movements,  there  is  progressive  weakness,  with  cyanosis, 
death  occurring  in  from  twelve  to  forty-eight  hours.  In  the 
more  prolonged  cases  widespread  oedema  and  extensive  enlarge- 
ment of  lymphatic  glands  are  marked  features  ;  and  in  the  glands, 
especially  about  the  neck,  actual  necrosis  with  ulceration  may 
occur,  constituting  the  so-called  anthrax  carbuncles.  Such 
subacute  conditions  are  especially  found  among  horses,  which 
are  by  nature  not  so  susceptible  to  the  disease  as  cattle  and 
sheep. 

On  post  mortem  examination  of  an  ox  dead  of  anthrax,  the 
most  noticeable  feature — one  which  has  given  the  name  "  splenic 
fever "  to  the  disease — is  the  enlargement  of  the  spleen,  which 
may  be  two  or  three  times  its  natural  size.  It  is  of  dark-red 
colour,  and  on  section  the  pulp  is  very  soft  and  friable,  sometimes 
almost  diffluent.  A  cover-glass  preparation  may  be  made  from 
the  spleen  and  stained  with  watery  methylene-blue.  On  examina- 
tion it  will  be  found  to  contain  enormous  numbers  of  bacilli 


ANTHRAX  IN  ANIMALS  307 

mixed  with  red  corpuscles  and  leucocytes,  chiefly  lymphocytes 
and  the  large  mononucleated  variety  (Fig.  108).  Pieces  of  the 
organ  may  be  hardened  in  absolute  alcohol,  and  sections  cut  in 
paraffin.  These  are  best  stained  by  Gram's  method.  Micro- 
scopic examination  of  such  shows  that  the  structure  of  the  pulp 
is  considerably  disintegrated,  whilst  the  bacilli  swarm  throughout 
the  organ,  lying  irregularly  amongst  the  cellular  elements.  The 


FIG.  108. — Scraping  from  spleen  of  guinea-pig  dead  of  anthrax,  showing  the 

bacilli  mixed- with  leucocytes,  etc.     (Same  appearance  as  in  the  ox.) 

"  Corrosive  film  "  stained  with  carbol-thionin-blue.      x  1000. 

liver  is  enlarged  and  congested,  and  may  be  in  a  state  of  acute 
cloudy  swelling.  The  bacilli  are  present  in  the  capillaries 
throughout  the  organ,  but  are  not  so  numerous  as  in  the  spleen. 
The  kidney  is  in  a  similar  condition,  and  here  the  bacilli  are 
chiefly  found  in  the  capillaries  of  the  glomeruli,  which  often 
appear  as  if  injected  with  them.  The  lungs  are  congested  and 
may  show  catarrh,  whilst  bacilli  are  present  in  large  numbers 
throughout  the  capillaries,  and  may  also  be  found  in  the  air  cells, 
probably  as  the  result  of  rupture  of  the  capillaries.  The  blood 
throughout  the  body  is  usually  fluid  and  of  dark  colour. 


308  ANTHRAX 

The  lymphatic  system  generally  is  much  affected.  The 
glands,  especially  the  mediastinal,  mesenteric,  and  cervical 
glands,  are  enlarged  and  surrounded  by  oedematous  tissue,  the 
lymphatic  vessels  are  swollen,  and  both  glands  and  vessels  may 
contain  numberless  bacilli.  The  heart  may  be  in  a  state  of  cloudy 
swelling,  and  the  blood  in  its  cavities  contains  bacilli,  though  in 
smaller  numbers  than  that  in  the  capillaries.  The  intestines  are 
enormously  congested,  the  epithelium  more  or  less  desquamated, 
and  the  lumen  filled  with  a  bloody  fluid.  From  all  the  organs 
the  bacilli  can  be  easily  isolated  by  stroke  cultures  on  agar. 

It  is  important  to  note  the  existence  of  great  differences  in 
susceptibility  to  anthrax  in  different  species  of  animals.  Thus 
the  ox,  sheep  (except  those  of  Algeria,  which  only  succumb  to 
enormous  doses  of  the  bacilli),  guinea-pig,  and  mouse  are  all  very 
susceptible,  the  rabbit  slightly  less  so.  The  last  three  are  of 
course  most  used  for  experimental  inoculation.  We  have  no 
data  to  determine  whether  the  disease  occurs  among  these  in  the 
wild  state.  Less  susceptible  than  this  group  are  the  horse,  deer, 
goat,  in  which  the  disease  occurs  from  time  to  time' in  nature. 
Anthrax  also  occurs  epidemically  in  the  pig,  often  from  the 
ingestion  of  the  organs  of  other  animals  dead  of  the  disease.  It 
is,  however,  doubtful  if  all  cases  of  disease  in  the  pig  described 
on  clinical  grounds  as  anthrax  are  really  such,  and  a  careful 
bacteriological  examination  is  always  advisable.  The  human 
subject  may  be  said  to  occupy  a  medium  position  between  the 
highly  susceptible  and  the  relatively  immune  animals.  The 
white  rat  is  highly  immune  to  the  disease,  while  the  brown  rat 
is  susceptible.  Adult  carnivora  are  also  very  immune,  and  the 
birds  and  amphibia  are  in  the  same  position. 

With  these  differences  in  susceptibility  there  are  also  great 
variations  in  the  pathological  effects  produced  in  the  natural  or 
artificial  disease.  This  is  especially  the  case  when  we  consider 
the  distribution  of  the  bacilli  in  the  bodies  of  the  less  susceptible 
animals.  Instead  of  the  widespread  occurrence  described  above, 
they  may  be  confined  to  the  point  where  they  first  gained  access 
to  the  body  and  the  lymphatic  system  in  relation  to  it,  or  may 
be  only  very  sparsely  scattered  in  organs  such  as  the  spleen 
(which  is  often  not  enlarged),  the  lungs,  or  kidneys.  Neverthe- 
less the  cellular  structure  of  the  organs  even  in  such  a  case  may 
show  changes,  a  fact  which  is  important  when  we  consider  the 
essential  pathology  of  the  disease. 

Experimental  Inoculation. — Of  the  animals  commonly  used 
in  laboratory  work,  white  mice  and  guinea-pigs  are  the  most 
susceptible  to  anthrax,  and  are  generally  used  for  test  inocula- 


ANTHRAX  IN   MAN  309 

tions.  If  a  small  quantity  of  anthrax  bacilli  be  injected  into  the 
subcutaneous  tissue  of  a  guinea-pig  a  fatal  result  follows,  usually 
within  two  days.  Post  mortem  around  the  site  of  inoculation  the 
tissues,  owing  to  intense  inflammatory  oedema,  are  swollen  and 
gelatinous  in  appearance,  small  haemorrhages  are  often  present, 
and  on  microscopic  examination  numerous  bacilli  are  seen. 
The  internal  organs  show  congestion  and  cloudy  swelling,  with 


FIG.  109. — Portion  of  kidney  of  a  guinea-pig  dead  of  anthrax,  showing  the 

bacilli  in  the  capillaries,  especially  of  the  glomerulus. 
Paraffin  section  ;  stained  by  Gram's  method  and  Bismarck-brown.      x  300. 

sometimes  small  haemorrhages,  and  their  capillaries  contain 
enormous  numbers  of  bacilli,  as  has  already  been  described  in 
the  case  of  the  ox  (Fig.  109) ;  the  spleen  also  shows  a  corre- 
sponding condition.  Highly  susceptible  animals  may  be  infected 
by  being  made  to  inhale  the  bacilli  or  their  spores,  and  also  by 
being  fed  with  spores,  a  general  infection  rapidly  occurring  by 
both  methods. 

Anthrax  in  the  Human  Subject. — As  we  have  noted,  man 
occupies  a  middle  position  in  the  scale  of  susceptibility  to 
anthrax.  It  is  always  communicated  to  him  from  animals,  and 


310  ANTHRAX 

usually  is  seen  among  those  whose  trade  leads  them  to  handle 
the  carcases  or  skins  of  animals  which  have  died  of  the  disease. 
It  occurs  in  two  principal  forms,  the  main  difference  between 
which  is  due  to  the  site  of  entrance  of  the  organism  into  the 
body.  In  one,  the  path  of  entrance  is  through  cuts  or  abrasions 
in  the  skin,  or  through  the  hair  follicles.  A  local  condition 
called  a  "malignant  pustule"  develops,  which  may  lead  to  a 
general  infection.  This  variety  occurs  chiefly  among  butchers 
and  those  who  work  among  hides  (foreign  ones  especially).  In 
Britain  the  workers  of  the  latter  class  chiefly  liable  are  the  hide- 
porters  and  hide- workers  in  South-Eastern  London.  In  the  other 
variety  of  the  disease  the  site  of  infection  is  the  trachea  and 
bronchi,  and  here  a  fatal  result  almost  always  follows.  The 
cause  is  the  inhalation  of  dust  or  threads  from  wool,  hair,  or 
bristles,  which  have  been  taken  from  animals  dead  of  the  disease, 
and  which  have  been  contaminated  with  blood  or  secretions  con- 
taining the  bacilli,  these  having  afterwards  formed  spores.  This 
variety  is  often  referred  to  as  "  woolsorter's  disease,"  from  its 
occurring  in  the  centres  of  the  woolstapling  trade  (in  England, 
chiefly  in  Yorkshire),  but  it  also  is  found  in  places  where  there 
are  hair  and  brush  factories. 

(1)  Malignant  Pustule. — This  usually  occurs  on  the  exposed 
surfaces — the  face,  hands,  fore-arms,  and  back,  the  last  being  a 
common  site  among  hide-porters.  One  to  three  days  after 
inoculation  a  small  red  painful  pimple  appears,  soon  becoming  a 
vesicle,  which  may  contain  clear  or  blood-stained  fluid,  and  is 
rapidly  surrounded  by  an  area  of  intense  congestion.  Central 
necrosis  occurs  and  leads  to  the  malignant  pustule  proper,  which 
in  its  typical  form  appears  as  a  black  eschar  often  surrounded 
by  an  irregular  ring  of  vesicles,  these  in  turn  being  surrounded 
by  a  congested  area.  From  this  pustule  as  a  centre  subcutaneous 
oedema  spreads,  especially  in  the  direction  of  the  lymphatics; 
the  neighbouring  glands  are  enlarged.  There  is  fever  with 
general  malaise.  On  microscopic  section  of  the  typical  pustule, 
the  central  eschar  is  noticed  to  be  composed  of  necrosed  tissue 
and  degenerating  blood  cells ;  the  vesicles  are  formed  by  the 
raising  of  the  stratum  corneum  from  the  rete  Malpighi.  Beneath 
them  and  in  their  neighbourhood  the  cells  of  the  latter  are 
swollen  and  cedematous,  the  papillae  being  enlarged  and  flattened 
out  and  infiltrated  with  inflammatory  exudation,  which  also 
extends  beneath  the  centre  of  the  pustule.  In  the  tissue  next 
the  eschar  necrosis  is  commencing.  The  subcutaneous  tissue  is 
also  oedematous,  and  often  infiltrated  with  leucocytes.  The 
bacilli  exist  in  the  periphery  of  the  eschar  and  in  the  neigh- 


ANTHRAX  IN   MAN  311 

bouring  lymphatics,  and,  to  a  certain  extent,  in  the  vesicles.  It 
is  very  important  to  note  that  widespread  oedema  of  a  limb, 
enlargement  of  neighbouring  glands,  and  fever  may  occur  while 
the  bacilli  are  still  confined  to  the  immediate  neighbourhood  of 
the  pustule.  Sometimes  the  pathological  process  goes  no  further, 
the  eschar  becomes  a  scab,  the  inflammation  subsides,  and 
recovery  takes  place.  In  the  majority  of  cases,  however,  if  the 
pustule  be  not  excised,  the  oedema  spreads,  invasion  of  the  blood 
stream  may  occur,  and  the  patient  dies  with,  in  a  modified 
degree,  the  pathological  changes  detailed  with  regard  to  the 
acute  disease  in  cattle.  In  man  the  spleen  is  usually  not  much 
enlarged,  and  the  organs  generally  contain  few  bacilli.  The 
actual  cause  of  death  is  therefore  the  absorption  of  toxins.  It 
may  here  be  said  that  early  excision  of  an  anthrax  pustule, 
especially  when  it  is  situated  in  the  extremities,  is  followed,  in  a 
large  proportion  of  cases,  by  recovery. 

(2)  Woolsorter's   Disease. — The   pathology  of   this   affection 
was  worked  out  in  this  country  especially  by  Greenfield.     The 
local  lesion  is  usually  situated  in  the  lower  part  of  the  trachea  or 
in  the  large  bronchi,  and  is  in  the  form  of  swollen  patches  in 
the  mucous  membrane  often  with  hemorrhage  into  them,     The 
tissues  are  cedematous,  and  the  cellular  elements  are  separated, 
but  there  is  usually  little  or  no  necrosis.     There  is  enormous 
enlargement   of   the    mediastinal    and    bronchial    glands,    and 
haemorrhagic  infiltration  of  the  cellular  tissue  in   the  region. 
There  are  pleural  and  pericardial  effusions,  and   hsemorrhagic 
spots  occur  beneath  the  serous  membranes.      The  lungs  show 
collapse  and  oedema.     There  may  be  cutaneous  oedema  over  the 
chest  and  neck,  with  enlargement  of  glands,  and  the  patient 
rapidly  dies  with  symptoms  of  pulmonary  embarrassment,  and 
with  a  varying  degree  of  pyrexia.     It  is  to  be  noted  that  in  such 
cases,   though  numerous  bacilli  are    present    in    the  bronchial 
lesions,   in  the   lymphatic   glands   and  affected  tissues  in  the 
thorax,  comparatively  few  may  be  present  in  the  various  organs, 
such  as  the  kidney,  spleen,  etc.,  and  sometimes  it  may  be  im- 
possible to  find  any. 

(3)  Infection  occasionally  takes  place  through  the  intestine, 
probably  by   ingestion  of    spores  as   in  the   case   of   animals ; 
but  this  condition  is  rare.     In  such  cases  there  is  a  local  lesion 
in  the  intestinal  mucous  membrane,  of  similar  nature  to  that  in 
the  bronchial  form,  the  central  parts  of  the  hsemorrhagic  areas 
being,  however,  sometimes  necrotic  and  yellowish,  and  there  is  a 
corresponding  affection  of  the  mesenteric  glands.     In  a  case  of 
this  kind,  recently  recorded  by  Teacher  hsemorrhagic  meningitis, 


312  ANTHRAX 

associated  with  the  presence  of   the  bacilli  in  large  numbers, 
occurred  as  a  complication. 

The  Toxins  of  the  Bacillus  Anthracis. — Various  theories 
were  formerly  held  as  to  the  mode  in  which  the  anthrax  bacillus 
produces  its  effects.  One  of  the  earliest  was  the  mechanical, 
according  to  which  it  was  supposed  that  the  serious  results  were 
produced  by  extensive  blocking  of  the  capillaries  in  the  various 
organs  by  the  bacilli.  According  to  another,  it  was  supposed 
that  the  bacilli  used  up  the  oxygen  of  the  blood,  thus  leading  to 
starvation  of  the  tissues.  The  discovery  of  definite  toxins  which 
accounted  for  the  pathogenic  effects  of  certain  bacteria  led  to 
such  bodies  being  sought  for  in  connection  with  the  anthrax 
bacillus.  Among  other  workers,  Sidney  Martin  investigated  this 
subject.  This  observer  used  alkali-albumin  on  which  to  grow 
the  bacillus,  this  medium  approaching  most  closely  to  the 
environment  of  the  latter  when  growing  in  the  animal  body. 
From  cultures  in  this  medium,  concentrated  by  evaporation 
either  at  100°  C.  or  in  vacuo  at  35°  to  45°  C.,  there  were 
isolated  proto-albumose,  deutero-albumose,  and  traces  of  peptone. 
The  albumoses  differed  from  those  which  occur  in  ordinary 
digestion,  in  being  strongly  alkaline  in  their  reaction.  This 
alkalinity,  Martin  held,  was  due  to  traces  of  an  alkaloidal  body 
of  which  the  albumoses  were  the  precursors,  and  which  were 
formed  when  the  process  of  digestion  of  the  alkali-albumin  by 
the  bacillus  was  allowed  to  go  on  further.  By  the  albumoses 
and  the  alkaloid,  pathogenic  effects  were  produced  in  animals, 
closely  similar  to  those  produced  by  the  bacilli  themselves. 
Martin,  to  account  for  the  symptoms  of  the  disease,  considered 
that  the  fever  was  mostly  due  to  the  albumoses,  while  the 
oedema  and  congestion  were  due  to  the  alkaloid  which  acted  as  a 
local  irritant.  He  showed  that  prolonged  boiling  destroyed  the 
activity  of  the  albumoses,  but  not  that  of  the  alkaloid.  Further, 
from  the  body  fluids  of  animals  dead  of  anthrax  he  isolated 
poisonous  bodies  similar  to  those  produced  by  the  bacilli  growing 
in  this  artificial  medium.  Hankin  and  Wesbrook  arrived  at  the 
conclusion  that  the  bacillus  anthracis  produces  a  ferment  which, 
diffusing  out  into  the  culture  fluid,  elaborates  albumoses  from 
the  proteids  present  in  it.  The  bacilli  also  produce  albumoses 
directly  without  the  intervention  of  a  ferment.  Marmier,  after 
cultivating  the  b.  anthracis  in  peptone  solution  containing 
certain  salts,  removed  all  the  albumoses  from  the  resultant 
liquid,  and  from  them,  either  by  dialysis  or  extraction  with 
glycerin,  isolated  a  body  which  gave  no  reactions  of  albuminoid 
matter,  peptone,  propeptone,  or  alkaloid.  This  he  considered  the 


SPREAD   OF   THE   DISEASE   IN   NATURE      313 

toxin.  It  killed  animals  susceptible  to  anthrax  by  a  sort  of 
cachexia,  and  in  suitably  small  doses  could  be  used  to  immunise 
them  against  subsequent  inoculation  with  virulent  bacilli.  It 
was  chiefly  retained  within  the  bacilli  when  these  were  growing 
in  the  most  favourable  conditions.  Unlike  the  toxins  of 
tetanus  and  diphtheria,  and  unlike  ferments,  it  was  not 
destroyed  by  heating  to  110°  C.  The  toxin  produced  by  the 
b.  anthracis  growing  in  a  fluid  medium  remains  intimately 
associated  with  the  bacterial  protoplasm,  as  such  cultures  when 
filtered  are  relatively  non-toxic. 

It  cannot  be  said  that  great  light  has  been  thrown  on  the 
pathology  of  the  disease  by  these  researches.  The  effects  of 
infection  by  the  b.  anthracis  are  those  shared  by  all  other 
organisms  producing  inflammation,  the  tendency  to  •  cedema 
production  of  an  unwonted  degree  being  the  chief  special 
feature  and  one  with  reference  to  which  Martin's  work  may  be 
important.  That  toxic  effects  do  occur  in  anthrax  is  undoubted, 
for  frequently,  while  the  bacilli  are  still  locally  confined,  there 
may  occur  pyrexia  and  cedema  spreading  widely  beyond  the 
pustule,  but  we  have  no  definite  information  as  to  how  these 
effects  are  produced.  In  this  disease  we  are  probably  dealing 
with  another  example  of  the  action  of  intracellular  toxins, 
regarding  which,  as  in  other  cases,  little  is  known. 

The  Spread  of  the  Disease  in  Nature. — We  have  seen  that 
the  b.  anthracis  rarely,  if  ever,  forms  spores  in  the  body,  and  if 
the  bacilli  could  be  confined  to  the  blood  and  tissues  of  carcases 
of  animals  dying  of  the  disease,  it  is  certain  that  anthrax  in  an 
epidemic  form  would  rarely  occur.  For  it  has  been  shown  by 
many  observers  that  in  the  course  of  the  putrefaction  of  such  a 
carcase  the  anthrax  bacilli  rapidly  die  out,  and  that  after  ten 
days  or  a  fortnight  very  few  remain.  But  it  must  be  remembered 
that  while  still  alive,  an  animal  is  shedding  into  the  air  by  the 
bloody  excretions  from  the  mouth,  nose,  and  bowel,  myriads  of 
bacilli  which  may  rapidly  spore,  and  thus  arrive  at  a  very  re- 
sistant stage.  These  lie  on  the  surface  of  the  ground  and  are 
washed  off  by  surface  water.  At  certain  seasons  of  the  year  the 
temperature  is,  however,  sufficiently  high  to  permit  of  their 
germination,  and  also  of  their  multiplication,  as  they  can  un- 
doubtedly grow  on  the  organic  matter  which  occurs  in  nature. 
They  can  again  form  spores.  It  is  in  the  condition  of  spores 
that  they  are  dangerous  to  susceptible  animals.  In  the  bacillary 
stage,  if  swallowed,  they  will  be  killed  by  the  acid  gastric  con- 
tents; but  as  spores  they  can  pass  uninjured  through  the 
stomach,  and  gaining  an  entrance  into  the  intestine,  infect  its 


314  ANTHRAX 

wall,  and  ultimately  reach,  and  multiply  in  the  blood.  It  is 
known  that  in  the  great  majority  of  cases  of  the  disease  in  sheep 
and  oxen,  infection  takes  place  thus  from  the  intestine.  It  was 
thought  by  Pasteur  that  worms  were  active  agents  in  the  natural 
spread  of  the  disease  by  bringing  to  the  surface  anthrax  spores. 
Koch  made  direct  experiments  on  this  point,  and  could  get  no 
evidence  that  such  was  the  case.  He  thinks  it  much  more 
probable  that  the  recrudescence  of  epidemics  in  fields  where 
anthrax  carcases  have  been  buried  is  due  to  persistence  of 
spores  on  the  surface  which  has  been  infected  by  the  cattle  when 
alive. 

The  Disposal  of  the  Carcases  of  Animals  dead  of  Anthrax.— It  is  ex- 
tremely important  that  anthrax  carcases  should  be  disposed  of  in  such  a 
way  as  to  prevent  their  becoming  future  sources  of  infection.  If  anthrax 
be  suspected  as  the  cause  of  death  no  post  mortem  examination  should  be 
made,  but  only  a  small  quantity  of  blood  removed  from  an  auricular 
vein  for  bacteriological  investigation.  If  such  a  carcase  be  now  buried 
in  a  deep  pit  surrounded  by  quicklime,  little  danger  of  infection  will  be 
run.  The  bacilli  being  confined  within  the  body  will  not  spore,  and 
will  die  during  the  process  of  putrefaction.  The  danger  of  sporulation 
taking  place  is,  of  course,  much  greater  when  an  animal  has  died  of  an 
unknown  disease  which  on  post  mortem  examination  has  proved  to  be 
anthrax,  but  similar  measures  for  burial  must  be  here  adopted.  In  some 
countries  anthrax  carcases  are  burned,  and  this,  if  practicable,  is  of 
course  the  best  means  of  treating  them.  The  chief  source  of  danger  to 
cattle  subsequently,  however,  proceeds  from  the  infection  of  fields,  yards, 
and  byres  with  the  offal  and  the  discharge  from  the  mouths  of  anthrax 
animals.  All  material  that  can  be  recognised  as  such  should  be  burned 
along  with  the  straw  in  which  the  animals  have  lain.  The  stalls  or 
buildings  in  which  the  anthrax  cases  have  been  must  be  limewashed. 
Needless  to  say,  the  greatest  care  must  be  taken  in  the  case  of  men  who 
handle  the  animal  or  its  carcase  that  they  have  no  wounds  on  their 
persons,  and  that  they  thoroughly  disinfect  themselves  by  washing  their 
hands,  etc.,  in  1  to  1000  solution  of  corrosive  sublimate,  and  that  all 
clothes  soiled  with  blood,  etc.,  from  anthrax  animals  be  thoroughly 
boiled  or  steamed  for  half  an  hour  before  being  washed. 

The  Immunising  of  Animals  against  Anthrax. — Having 
ascertained  that  there  was  ground  for  believing  that  in  cattle 
one  attack  of  anthrax  protected  against  a  second,  Pasteur  (in 
the  years  1880-82)  elaborated  a  method  by  which  a  mild  form 
of  the  disease  could  be  given  to  animals,  which  rendered 
harmless  a  subsequent  inoculation  with  virulent  bacilli.  He 
found  that  the  continued  growth  of  anthrax  bacilli  at  42°  to 
43°  C.  caused  them  to  lose  their  capacity  of  producing  spores, 
and  also  gradually  to  lose  their  virulence,  so  that  after  twenty- 
four  days  they  could  no  longer  kill  either  guinea-pigs,  rabbits, 
or  sheep.  Such  cultures  constituted  his  premier  vaccin,  and 


IMMUNISATION  AGAINST  ANTHRAX         315 

protected  against  the  subsequent  inoculation  with  bacilli  which 
had  been  grown  for  twelve  days  at  the  same  temperature,  and 
the  attenuation  of  which  had  therefore  not  been  carried  so  far. 
The  latter  constituted  the  deuxieme  vaccin.  It  was  further 
found  that  sheep  thus  twice  vaccinated  now  resisted  inoculation 
with  a  culture  which  usually  would  be  fatal.  The  method  was 
to  inoculate  a  sheep  on  the  inner  side  of  the  thigh  by  the 
subcutaneous  injection,  from  a  hypodermic  syringe,  of  about 
five  drops  of  the  premier  vaccin ;  twelve  days  later  to  again 
inoculate  with  the  deuxieme  vaccin  ;  fourteen  days  later  an 
ordinary  virulent  culture  was  injected  without  any  ill  result. 
This  method  was  applicable  also  to  cattle  and  horses,  about 
double  the  dose  of  each  vaccine  being  here  necessary.  Extended 
experiments  in  France  generally  confirmed  earlier  results,  and 
the  method  was,  before  long,  used  to  mitigate  the  disease,  which 
in  many  departments  was  endemic  and  a  very  great  scourge. 
Since  that  time  the  method  has  been  regularly  in  use.  It  is 
difficult  to  arrive  at  a  .  certain  conclusion  as  to  its  merits. 
Undoubtedly  a  certain  number  of  animals  die  of  anthrax  either 
after  the  first  or  second  vaccination,  or  during  the  year  following 
vaccination.  At  the  end  of  a  year  the  immunity  is  lost  in 
about  40  per  cent  of  the  animals  vaccinated ;  and  thus  to  be 
permanently  efficacious  the  process  would  have  to  be  repeated 
every  year.  Further,  the  immunity  is  much  higher  in  degree 
if,  after  the  first  and  second  vaccinations,  an  inoculation  with 
virulent  anthrax  is  performed.  Everything  being  taken  into 
account,  however,  there  is  no  doubt  that  the  mortality  from 
natural  anthrax  is  much  diminished  by  this  system. 

During  the  twelve  years  1882-93  3,296,815  sheep  were  vaccinated, 
with  a  mortality,  either  after  the  first  or  second  vaccination,  or  during 
the  subsequent  twelve  months,  of  '94  per  cent,  as  contrasted  with  the 
ordinary  mortality  in  all  the  flocks  of  the  districts  of  10  per  cent. 
During  the  same  time  438,824  cattle  were  vaccinated,  with  a  mortality 
of  '34  per  cent,  as  contrasted  with  a  probable  mortality  of  5  per  cent  if 
they  had  been  unprotected. 

The  immunisation  of  animals  against  anthrax  has  always 
been  found  to  be  a  difficult  proceeding.  The  most  usual 
technique  has  been  to  commence  with  Pasteur's  vaccines,  and  to 
follow  these  by  careful  dosage  with  virulent  cultures.  Marchoux 
in  this  way  produced  immunity,  and  found  that  the  serum  of 
immune  animals  had  a  certain  degree  of  protective  and  curative 
action.  The  most  successful  attempts  in  this  direction  have 
been  those  of  Sclavo  and  of  Sobernheim.  The  former  observer, 
after  trying  various  animals,  came  to  the  conclusion  that  the 


316  ANTHRAX 

ass  was  the  most  suitable.  He  first  employed  a  method  similar 
to  that  of  Marchoux;  later,  however,  after  noting  the  effects 
of  the  serum  of  an  animal  so  immunised,  he  commenced  the 
immunisation  by  injecting  5  to  15  c.c.  of  this  serum  along  with 
a  slightly  attentuated  culture  of  the  bacilli.  A  few  days  later 
this  was  followed  up  with  injections  of  virulent  cultures  which 
could  now  be  periodically  introduced  for  many  months,  and  a 
high  degree  of  immunity  resulted.  What  was  even  more 
important,  the  serum  of  such  an  animal  had  strongly  protective 
and  curative  properties.  It  has  been  extensively  used  in  the 
treatment  of  anthrax  in  man.  In  a  case  of  malignant  pustule 
30  to  40  c.c.  are  injected  in  quantities  of  10  c.c.  into  the 
abdominal  wall,  and  if  necessary  the  injection  is  repeated  on  the 
following  day.  In  cases  treated  by  Sclavo  himself  the  serum  is 
alone  employed,  and  its  action  is  not  aided  by  the  excision 
of  the  pustule  usually  practised.  The  results  obtained  have  been 
very  good, — Sclavo,  out  of  164  cases,  had  only  ten  deaths  or 
about  a  fourth  of  the  ordinary  mortality  in  Italy.  Sobernheim 
independently  elaborated  an  almost  identical  method  of  com- 
bining passive  with  active  immunisation  for  the  obtaining  of  a 
powerful  anti-serum,  and  he  has  used  this  for  the  protective 
inoculation  of  cattle.  The  technique  is  to  inject  the  serum  into 
one  side  of  the  neck  or  into  one  thigh  and  the  culture  (Pasteur's 
second  vaccine)  into  the  other  side ;  the  doses  given  are  for 
cattle  or  horses  5  c.c.  of  serum  and  '5  c.c.  culture,  and  for  sheep 
4  c.c.  of  serum  and  '25  c.c.  culture.  The  method  has  been  widely 
used  in  Germany  and  in  Brazil,  and  its  originator  claims  as  its 
advantages  simplification  of  application,  in  that  one  operation 
instead  of  two  is  sufficient,  less  risk  of  death  following  the 
immunisation  procedure,  and  higher  degree  and  more  lasting 
character  of  the  immunity  resulting.  Whether  this  method  is 
really  more  efficient  than  that  of  Pasteur  future  experience  will 
show,  but  it  might  be  preferable  for  developing  protection  in 
herds  at  a  time  when  an  epidemic  was  raging.  During  the 
development  of  active  immunity  it  is  likely  in  every  case  (see 
Immunity)  that  there  is  a  period  of  increased  susceptibility  to  the 
disease.  Such  a  period  would  be  more  likely  to  occur  with  the 
Pasteur  method  than  with  the  Sobernheim  procedure,  where  the 
presence  in  the  animal's  body  of  the  protective  serum  might  tide 
it  over  the  stage  when  the  action  of  the  vaccine  was  lowering 
its  resistance. 

The  effects  of  the  b.  anthracis  have  been  much  studied  with 
a  view  to  the  shedding  of  light  on  the  processes  obtaining  in 
resistance  and  the  development  of  immunity.  Many  puzzling 


METHODS   OF  EXAMINATION  317 

facts  have  long  been  known;  for  example,  in  the  dog,  which 
shows  great  natural  resistance,  the  serum  has  little  if  any 
bactericidal  action,  while  in  the  susceptible  rabbit  there  is 
present  a  serum  capable  of  killing  the  organism.  Such  observa- 
tions have  hitherto  been  without  explanation.  Again  the 
properties  of  the  serum  of  immune  animals  have  been  much 
discussed.  Sobernheim  and  others  have  been  unable  to  detect 
in  it  any  trace  of  special  bactericidal  action.  Sclavo  found  that 
the  serum  when  heated  to  55°  C.  did  not  lose  its  protective 
properties ;  as  the  serum  might  have  been  complemented  (see 
Immunity)  by  the  serum  of  the  animal  into  which  it  was  injected, 
he  simultaneously  introduced  an  anti-complementary  serum  and 
found  that  the  heated  serum  was  still  effectual.  From  this  he 
deduces  that  in  the  action  of  the  serum  substances  of  the  nature 
of  immune  body  and  complement  are  not  concerned.  Many 
have  thought  that  the  serum  had  a  stimulating  effect  on  the 
leucocytes,  but  Cler  has  brought  forward  ground  for  supposing 
that  its  effect  is  a  sensitising  one  on  the  bacteria,  and  that  thus 
the  effects  are  to  be  traced  to  opsonic  action.  With  regard  to 
the  formation  of  the  protective  substances,  it  is  stated  that  the 
spleen  and  bone-marrow  are  richer  in  these  than  the  blood  fluids. 
In  this  connection  an  interesting  fact  may  be  mentioned,  namely, 
that  Roger  and  Gamier  found  evidence  of  the  liver  and  spleen 
having  special  capacities  for  killing  anthrax  bacilli ;  an  otherwise 
fatal  dose  could  be  introduced  into  the  portal  vein  or  the  splenic 
artery  without  causing  death. 

Methods  of  Examination. — These  include  (a)  microscopic 
examination ;  (b)  the  making  of  cultures ;  and  (c)  test  in- 
oculations. 

(a)  Microscopic  Examination.  —  In  a  case  of  suspected 
malignant  pustule,  film  preparations  should  be  made  from  the 
fluid  in  the  vesicles  or  from  a  scraping  of  the  incised  or  excised 
pustule,  and  stained  with  a  watery  solution  of  methylene-blue 
and  also  by  Gram's  method.  By  this  method  practically  con- 
clusive evidence  may  be  obtained ;  but  sometimes  the  result  is 
doubtful,  as  the  bacilli  may  be  very  few  in  number.  In  all 
cases  confirmatory  evidence  should  be  obtained  by  culture. 
Occasionally  bacilli  are  so  scanty  that  both  film  preparations 
made  from  different  parts  and  even  cultures  may  give  negative 
results,  and  yet  a  few  bacilli  may  be  found  when  a  section  of 
the  pustule  is  examined.  It  should  be  noted  that  the  greatest 
care  ought  to  be  taken  in  manipulating  a  pustule  before  excision, 
as  the  diffusion  of  the  bacilli  into  the  surrounding  tissues  may 
be  aided  and  the  condition  greatly  aggravated.  The  examination 


318  ANTHRAX 

of  the  blood  in  cases  of  anthrax  in  man  usually  gives  negative 
results,  with  the  exception  of  very  severe  cases,  when  a  few 
bacilli  may  be  found  in  the  blood  shortly  before  death,  though 
even  then  they  may  be  absent. 

(6)  Cultivation. — A  small  quantity  of  the  material  used  for 
microscopic  examination  should  be  taken  on  a  platinum  needle, 
and  successive  strokes  made  on  agar  tubes,  which  are  then 
incubated  at  37°  C.  At  the  end  of  twenty-four  hours  anthrax 
colonies  will  appear,  and  can  be  readily  recognised  from  their 
wavy  margins  by  means  of  a  hand  lens.  They  should  also  be 
examined  microscopically  by  means  of  film  preparations. 

(c)  Test  Inoculations. — A  little  of  the  suspected  material 
should  be  mixed  with  some  sterile  bouillon  or  water,  and 
injected  subcutaneously  into  a  guinea-pig  or  mouse,  or  it  may 
be  introduced  into  the  subcutaneous  tissue  by  means  of  a  seton. 
If  anthrax  bacilli  are  present,  the  animal  usually  dies  within 
two  days,  with  the  changes  in  internal  organs  already  described. 


CHAPTER   XIV. 

TYPHOID  FEVER— BACILLI  ALLIED  TO  THE 
TYPHOID  BACILLUS. 

OTHER     NAMES. ENTERIC     FEVER  :      GASTRTC      FEVER.         GERMAN, 

TYPHUS     ABDOMINALIS  :     ABDOMINALTYPHUS  I     UNTERLEIBS- 
TYPHUS.       FRENCH,    LA    FlfcVRE    TYPHOIDE. 

Introductory.  —  The  organism  now  known  as  the  bacillus 
typhosus  was  first  described  in  1880-1  by  Eberth,  who  observed 
its  microscopic  appearances  in  the  intestinal  ulcers  and  in  the 
spleen  in  cases  of  typhoid  fever.  It  was  first  isolated  (from 
the  spleen)  in  1884  by  Gaffky,  and  its  cultural  characters  were 
then  investigated.  In  1885  Escherich  observed  a  bacillus,  now 
known  as  the  bacillus  coli  communis,  which  occurs  in  the  normal 
intestine  and  which  both  microscopically  and  culturally  closely 
resembles  the  typhoid  bacillus.  Ordinarily  the  b.  coli  is  no 
doubt  a  harmless  saprophyte,  but  under  experimental  conditions 
in  animals  and  also  naturally  in  man  it  may  manifest  pathogenic 
properties.  Investigation  has  shown  that  these  two  bacilli 
belong  to  a  widespread  group  of  organisms  isolated  from  various 
disease  conditions,  which  all  bear  close  resemblances  to  one 
another  and  whose  differentiation  is  often  a  matter  of  consider- 
able difficulty.  Other  members  of  this  group  are  the  para- 
typhoid bacillus,  the  organism  of  bacillary  dysentery,  the  b. 
enteritidis  of  Gaertner,  the  psittacosis  bacillus,  and  the  bacillus 
of  hog  cholera. 

Bacillus  Typhosus.— Microscopic  Appearances. — It  is  some- 
times difficult  to  find  the  typhoid  bacilli  in  the  organs  of  a 
typhoid  patient.  Numerous  sections  of  different  parts  of  a 
spleen,  for  example,  may  be  examined  before  a  characteristic 
group  is  found.  The  best  tissues;  for  examination  are  a  Peyer's 
patch  where  ulceration  has  not  yet  commenced  or  where  it  is 

319 


320 


TYPHOID   FEVER 


just  commencing,  the  spleen,  the  liver,  or  a  mesenteric  gland. 
The  spleen  and  liver  are  better  than  the  other  tissues  named,  as 
in  the  latter  the  presence  of  the  b.  coli  is  more  frequent.  From 
scrapings  of  such  solid  organs  dried  films  may  be  prepared  and 
stained  for  a  few  minutes  in  the  cold  by  any  of  the  strong 
staining  solutions,  e.g.  with  carbol-thionin-blue,  or  with  Ziehl- 

Neelsen  carbol  -  fuchsin 
diluted  with  five  parts 
of  distilled  water.  As  a 
rule  decolorising  is  not 
necessary.  For  the  proper 
observation  of  the  ar- 
rangement of  the  bacilli 
in  the  tissues,  paraffin 
sections  should  be  pre- 
pared and  stained  in 
carbol-thionin-blue  for  a 
fewminutes,  orinLofner's 
methylene-blue  for  one 
or  two  hours.  The  bacilli 
take  up  the  stain  some- 
what slowly,  and  as  they 

FIG.  110. — A  large  clump  of  typhoid  bacilli  ,,    "     .,.        ^.,         ,,     ,     ]. 

in  a  spleen.     The  individual  bacilli   are  the  aniline-Oil  method  of 

only  seen  at  the  periphery  of  the  mass,  dehydration  may  be  used 

(In    this    spleen    enormous    numbers    of  with  advantage   (vide  p. 

typhoid  bacilli  were  shown  by  cultures  to  93^      jn    such    prepara_ 

tions  the  characteristic 
appearance  to  be  looked 
for  is  the  occurrence  of 

groups  of  bacilli  lying  between  the  cells  of  the  tissue  (Fig.  110). 
The  individual  bacilli  are  2  //,  to  4  //,  long,  with  somewhat  oval 
ends,  and  '5  /x  in  thickness.  Sometimes  filaments  8  /JL  to  10  ft 
long  may  be  observed,  though  they  are  less  common  than  in 
cultures.  It  is  evident  that  one  of  the  short  oval  forms  may 
frequently  in  a  section  be  viewed  endwise,  in  which  case  the 
appearance  will  be  circular.  This  appearance  accounts  for  some, 
at  least,  of  the  coccus-like  forms  which  have  been  described. 
The  bacilli  are  decolorised  by  Gram's  method. 

Isolation  and  Appearances  of  Cultures. — To  grow  the 
organism  artificially  it  is  best  to  isolate  it  from  the  spleen,  as  it 
exists  there  in  greater  numbers  than  in  the  other  solid  organs, 
and  may  be  the  sole  organism  present  even  some  time  after 
death.  The  spleen  is  removed  whole,  and  a  portion  of  its 


be  present  in  a  practically  pure  condition. ) 
Paraffin  section  ;  stained  with  carbol-thionin- 
blue.      x  500. 


ISOLATION  AND  APPEARANCES  OF  CULTURES     321 


capsule  is  seared  with  a  cautery  to  destroy  all  superficial  con- 
taminating organisms.  A  small  incision  is  made  into  the  organ 
with  a  sterile  knife,  a  little  of  the  pulp  removed  by  a  platinum 
needle,  and  agar  or  gelatin  plates  are  prepared,  or  successive 
strokes  made  on  agar  tubes.  On  the  agar  media  the  growths 
are  visible  after  twenty-four  hours'  incubation  at  37°  C.  On 
agar  plates  the  superficial  colonies  are  thin  and  film-like,  circular 
or  slightly  irregular  at  the  margins,  dull  white  by  reflected  light, 
bluish-grey  by  transmitted  light.  Colonies  in  the  substance  of 
the  agar  are  small,  and  appear  as  minute  round  points.  When 
viewed  under  a  low  ob- 
jective, the  surface 
colonies  are  found  to  be 
very  transparent  (requir- 
ing a  small  diaphragm 
for  their  definition),  finely 
granular  in  appearance, 
and  with  a  very  coarsely 
crenated  and  well-defined 
margin .  The  deep  colonies 
are  usually  spherical, 
sometimes  lenticular  in 
shape,  and  are  smooth  or 
finely  granular  on  the 
surface,  and  more  opaque 
than  the  superficial 

colonies.       On     making 

FIG.  111.— Typhoid    bacilli,    from    a 

culture  on  agar,  showing  some  filamentous 

forms. 

present  the   same   micro-    Stained  with  weak  carbol-fuchsin.      x    1000. 
scopic  appearances  as  are 

observed  in  preparations  from  solid  organs,  except  that  there 
may  be  a  greater  number  of  the  longer  forms  which  may 
almost  be  called  filaments  (Fig.  111).  The  same  is  true  of  films 
made  from  young  gelatin  colonies.  Sometimes  the  diversity  in 
the  length  of  the  bacilli  is  such  as  to  throw  doubt  on  the  purity 
of  the  culture.  Its  purity,  of  course,  can  be  readily  tested  by 
preparing  plates  from  it  in  the  usual  way.  As  a  general  rule  in 
a  young  (twenty-four  to  forty-eight  hours  old)  colony,  grown  at 
a  uniform  temperature,  the  bacilli  are  plump,  and  the  protoplasm 
stains  uniformly.  In  old  cultures,  or  in  cultures  which  have 
been  exposed  to  changes  of  temperature,  the  protoplasm  stains 
only  in  parts ;  there  may  be  an  appearance  of  irregular  vacuola- 
tion  either  at  the  centre  or  at  the  ends  of  the  bacilli.  There 
21 


cover -glass  preparations, 
the  bacilli  are  found  to 


young 


322 


TYPHOID   FEVER 


is   no    evidence    that    spore -formation    occurs    in    the    typhoid 
bacillus. 

Motility. — In  hanging-drop  preparations  the  bacilli  are  found 
to  be  actively  motile.  The  smaller  forms  have  a  darting  or 
rolling  motion,  passing  quickly  across  the  field,  whilst  some  show 
rapid  rotatory  motion.  The  filamentous  forms  have  an  un- 
dulating or  serpentine  motion,  and  move  more  slowly.  Hanging- 
drop  preparations  ought  to  be  made  from  agar  or  broth  cultures 


Fia.  112. — Typhoid  bacilli,  from  a  young  culture  on  agar,  showing  flagella. 
Stained  by  Van  Ermengem's  method.      x  1000. 

not  more  than  twenty-four  hours  old.  In  older  cultures  the 
movements  are  less  active. 

Flagella. — On  being  stained  by  the  appropriate  methods  (vide 
p.  103)  the  bacilli  are  seen  to  possess  many  long  wavy  flagella 
which  are  attached  all  along  the  sides  and  to  the  ends  (Fig.  1 1 2). 
They  are  more  numerous,  longer,  and  more  wavy  than  those  of 
the  b.  coli. 

Characters  of  Cultures. — Stab  cultures  in  peptone  gelatin  give 
a  somewhat  characteristic  appearance.  On  the  surface  of  the 
medium  growth  spreads  outwards  from  the  puncture  as  a  thin 


APPEARANCES   OF   CULTURES 


323 


film  or  pellicle,  with  irregularly  wavy  margin  (Fig.  113,  A).  It 
is  semi-transparent  and  of  bluish-white  colour.  Ultimately  this 
surface  growth  may  reach  the  wall  of  the  tube.  Not  infrequently, 
however,  the  surface  growth  is  not  well  marked.  Along  the 
stab  there  is  an  opaque  whitish  line  of  growth,  of  finely  nodose 
appearance.  There  is  no  liquefaction  of  the  medium,  and  no 
formation  of  gas.  In  stroke  cultures  there  is  a  thin  bluish-white 


ABC 
FIG.  113. 

A.  Stab  culture  of  the  typhoid  bacillus  in  gelatin,  five  days'  growth. 

B.  Stroke  culture  of  the  typhoid  bacillus  on  gelatin,  six  days'  growth. 

C.  Stab  culture  of  the  bacillus  coli  in  gelatin,  nine  days'  growth  ;  the  gelatin  is  split 
in  its  lower  part  owing  to  the  formation  of  gas. 

film,  but  it  does  not  spread  to  such  an  extent  as  in  the  case  of 
the  surface  growth  of  a  stab  culture  (Fig.  113,  B).  In  gelatin 
plates  also  the  superficial  and  deep  colonies  present  correspond- 
ing differences.  The  former  are  delicate  semi-transparent  films, 
with  wavy  margin,  and  are  much  larger  than  the  colonies  in  the 
substance,  which  appear  as  small  round  points  (Fig.  114).  These 
appearances,  which  are  well  seen  on  the  third  or  fourth  day, 
resemble  those  seen  in  agar  plates,  as  already  described  in  the 
method  of  isolation ;  but  on  gelatin  the  surface  colonies  are 


324 


TYPHOID   FEVER 


rather  more  transparent  than  those  on  agar.     Their  characters, 
as  seen  under  a  low  power  of  the  microscope,  also  correspond. 

In  stroke  cultures  on  agar  there  is  a  bluish-grey  film  of 
growth,  with  fairly  regular  margins,  but  without  any  character- 
istic features.  This  film  is  loosely  attached  to  the  surface,  and 
can  be  easily  scraped  off. 

The  growth  on  potatoes  is  important.  For  several  days  (at 
incubation  temperature)  after  inoculation  there  is  apparently 
no  growth.  If  looked  at  obliquely,  the  surface  appears  wet, 
and  if  it  is  scraped  with  the  platinum  loop,  a  glistening  track 

is  left ;  a  cover-glass  pre- 
paration shows  numerous 
bacilli.  Later,  however, 
a  slight  pellicle  with  a 
dull,  somewhat  velvety 
surface  may  appear,  and 
this  may  even  assume 
a  brown  appearance. 
These  characteristic  ap- 
pearances are  only  seen 
when  a  fresh  potato  with 
an  acid  reaction  has  been 
used. 

In  bouillon  incubated 
at  37°  C.  for  twenty-four 
hours  there  is  simply  a 
FIG.  114. — Colonies  of  the  typhoid  bacillus   uniform       turbidity, 
(one  superficial  and  three  deep)  in  a  gelatin    Cover-glass  preparations 
plate.     Three  days    growth  at  room  tern-  ,      °£ 

perature.      x  15.  made    from   such    some- 

times  show  filamentous 
forms  of  considerable  length  without  apparent  segmentation. 

Conditions  of  Growth,  etc. — The  optimum  temperature  of  the 
typhoid  bacillus  is  about  37°  C.,  though  it  also  flourishes  well  at 
the  room  temperature.  It  will  not  grow  below  9°  C.  or  above 
42°  C.  Growth  takes  place  in  anaerobic  as  well  as  in  aerobic 
conditions.  Its  powers  of  resistance  correspond  with  those  of 
most  non-sporing  bacteria.  It  is  killed  by  exposure  for  half  an 
hour  at  60°  C.,  or  for  two  or  three  minutes  at  100°  C.  Typhoid 
bacilli  kept  in  distilled  or  in  ordinary  tap  water  have  usually 
been  found  to  be  dead  after  three  weeks  (Frankland). 

Bacillus  coli  communis. — This  bacillus  is  the  chief  organism 
present  in  the  small  intestine  in  normal  conditions,  and,  with 
many  other  bacteria,  it  also  inhabits  the  large  intestine.  During 
typhoid  fever,  and  other  pathological  conditions  affecting  the 


REACTIONS   OF   B.    TYPHOSUS   AND   B.    COLI     325 


intestines,  it  is  relatively  and  absolutely  enormously  increased 
in  the  latter  situation,  where  it  may  sometimes  be  almost  the 
only  bacillus  present.  Its  relations  to  various  suppurative  and 
inflammatory  conditions  are  described  in  the  chapter  on  Suppura- 
tion (p.  185).  Microscopically  it  has  the  same  appearances  and 
staining  reaction  as  the  typhoid  bacillus,  and  like  the  latter  also 
presents  variations  in  size,  though  it  is  usually  somewhat  shorter 
(Fig.  115).  It  is  motile,  and  possesses  lateral  flagella,  which, 
however,  are  fewer  in  number  and  somewhat  shorter  than  those 
of  the  typhoid  bacillus.  It  is  easily  isolated  from  the  stools 
of  men  and  animals  by 
any  of  the  ordinary 
methods.  After  twenty- 
four  hours'  incubation  at 
37°  C.  on  agar,  there  are 
large  superficial  colonies 
and  small  deep  colonies 
in  the  plates ;  to  the 
naked  eye  they  are 
denser  and  more  glisten- 
ing than  those  of  typhoid 
when  viewed  by  trans- 
mitted light,  and  rather  of 
a  brownish-white  colour. 
Under  a  low  objective 
the  colonies  again  appear 
denser  than  those  of  the 
typhoid  bacillus  and  more 
granular.  On  ordinary 
gelatin  and  agar  media 

the  appearances  are  similar  to  those  of  the  typhoid  bacillus, 
but  the  growth  is  whiter,  thicker,  and  more  opaque,  and  gives 
the  impression  of  having  greater  vigour.  In  the  case  of  gelatin 
stab  cultures  a  few  gas  bubbles  sometimes  develop  in  the 
medium  (Fig.  113,  C).  On  potatoes  in  forty-eight  hours  there 
is  a  distinct  film  of  growth  of  brownish  tint  and  moist-looking 
surface,  which  rapidly  spreads  and  becomes  thicker.  This  con- 
trasts very  markedly  with  the  colourless  film  of  the  b.  typhosus. 
The  Comparative  Culture  Reactions  of  the  B.  typhosus 
and  the  B.  coli. — The  importance  of  the  relationships  between 
the  b.  typhosus  and  the  b.  coli  has  caused  great  attention  to  be 
paid  to  their  biological  characters,  in  order  to  facilitate  the 
distinction  of  the  one  from  the  other.  Some  of  these  we  have 
already  noted.  Of  the  cultural  characters  the  growth  on 


FIG.  115. — Bacillus  coli  communis.     Film 

preparation  from  a  young  culture  on  agar. 

Stained  with  weak  carbol-fuchsin.      x  1000. 


326  TYPHOID   FEVER 

potatoes  is  the  most  important.  As  has  been  pointed  out  by 
Wathelet,  and  also  by  Klein,  differences  exist  in  the  growth  of 
the  two  bacilli  in  melted  gelatin.  A  gelatin  tube  is  inoculated, 
and  instead  of  being  kept  at  the  room  temperature,  is  placed  in 
the  incubator  at  37°  C.,  at  which  temperature  it  is  of  course 
fluid.  In  such  cultures,  in  the  case  of  the  b.  typhosus,  there  is 
a  general  turbidity  of  the  gelatin,  while  with  the  b.  coli  there 
are  large  flocculi  developed  which  float  on  the  surface.  It  is, 
however,  to  physiological  differences  between  the  bacilli,  rather 
than  to  morphological,  that  importance  is  to  be  attached.  In 
detailing  the  following  reactions  we  must  note  that  all  that  can 
be  said  is  that  under  certain  conditions  certain  effects  are  obtained. 
We  cannot  profess  to  know  the  principles  which  underlie  the 
occurrence  of  these  effects,  and  it  may  be  that  in  several 
apparently  diverse  reactions  the  same  biological  processes  are 
really  at  work. 

(1)  The  Fermentation  of  Sugars. — Of  these  one  of  the  most 
important  is  the  effect  on  lactose  as  first  pointed  out  by 
Chantemesse  and  Widal.  This  is  usually  demonstrated  by 
using  a  1  per  cent  solution  of  the  sugar  in  peptone-salt  solution 
placed  in  Durham's  tubes  (p.  76).  If  such  a  medium  be 
coloured  with  litmus  the  production  of  acid  and  gas  by  the 
b.  coli  can  easily  be  demonstrated.  Similar  changes  caused  by 
this  organism  can  also  be  observed  in  litmus  milk  and  in 
Petruschky's  litmus  whey. 

Chantemesse  and  Widal  first  showed  that  the  b.  typhosus 
does  not  act  on  lactose  in  bouillon  though  decolorisation  of  the 
litmus  may  occur.  It  may  be  stated  that  under  most  conditions 
of  making  the  test  an  acid  reaction  does  not  result  and  there  is 
never  any  formation  of  gas.  This  organism  is  said,  however,  to 
break  up  lactose  in  litmus  milk  and  in  litmus  whey  with  some 
acid  formation.  Much  would  thus  seem  to  depend  upon  what 
other  constituents  are  present  in  the  medium,  and  also,  it  may  be 
said,  on  its  initial  reaction. 

The  lactose  fermenting  power  of  the  b.  coli  is  of  the  greatest 
importance ;  and  if  MacConkey's  bile-salt  lactose  fluid  medium  be 
used,  this  organism  and  its  congeners  can  be  distinguished  from 
the  b.  typhosus,  b.  paratyphosus,  and  from  the  dysentery  bacilli 
(v.  infra),  none  of  whose  colonies  are  crimson  on  this  medium. 

The  effects  of  the  b.  coli  and  the  b.  typhosus  on  other  sugars 
is  also  of  great  importance.  As  media  to  which  the  sugars  may 
be  added,  either  peptone-salt  solution  or  MacConkey's  bile-salt 
media  are  used  (q.v.).  To  sum  up  the  general  results  it  may  be 
said  that  b.  coli  produces  acid  and  gas  in  bile-salt  glucose, 


REACTIONS   OF   B.    TYPHOSUS   AND   B.    COLI     327 

peptone-salt  glucose,  lactose  and  mannite,  but  not  in  cane  sugar,1 
while  the  b.  typhosus  produces  acid  without  gas  in  bile-salt 
glucose,  peptone-salt  glucose,  and  mannite,  but  not  in  lactose  or 
cane  sugar.  It  can  also  cause  similar  changes  in  arabinose, 
galactose,  and  fructose. 

Gas  production  by  the  b.  coli  can  also  be  demonstrated 
by  means  of  shake  cultures.  As  ordinary  bouillon  contains 
traces  of  glucose  it  is  best  to  use  peptone-salt  solution  to  which 
an  appropriate  sugar  has  been  added  and  which  has  been 
converted  into  a  solid  medium  with  10  per  cent  gelatine.  If 
such  a  medium  be  inoculated  in  the  fluid  condition,  shaken  and 
set  aside  till  growth  occurs,  small  bubbles  of  gas  will  form  all 
throughout  it.  In  ordinary  media  inoculated  with  the  b.  coli 
bubbles  of  gas  are  often  developed  along  the  needle  track. 

In  the  case  of  acid  production  by  the  b.  coli  or  b.  typhosus  in 
ordinary  media  the  acid  probably  comes,  as  has  been  said,  from 
the  glucose  developed  from  the  muscle  sugar,  but  there  may  also 
be  a  subsidiary  acid  formation  from  the  breaking  up  of  the 
proteid  elements. 

In  certain  members  of  the  coli-typhoid  group  it  has  been 
observed  that  in  such  media  as  litmus  milk  or  litmus  whey  an 
acid  reaction  may  be  first  produced,  and  this  may  be  followed 
after  a  few  days  by  the  formation  of  alkali,  and  in  certain  cases 
this  phenomenon  may  be  helpful  in  differentiating  the  species. 

Curdling  of  Milk  by  the  B.  coli. — This  probably  depends  on 
the  fermentation  of  the  lactose  of  the  milk  and  the  throwing 
down  of  the  casein  by  the  resulting  lactic  acid  ;  but  the  reaction 
may  be  a  more  complicated  one,  as  milk  can  be  curdled  by 
organisms  which  do  not  possess  acid-forming  properties.  In  any 
case  the  observation  of  the  reaction  is  important.  The  typhoid 
bacillus  produces  no  visible  change  in  milk. 

(2)  Action  on  Media  containing  Neutral- Red. — While,  as  will 
have  been  already  gathered,  neutral-red  is  used  as  an  indicator, 
there  is  some  evidence  that  an  actual  breaking  up  of  the 
substance  can  take  place  by  the  action  of  the  coli-typhoid  group  ; 
the  evidence  for  this  lies  in  the  fact  that  when  the  effects  of 
acid  formation  are  observed  the  tint  of  the  medium  cannot  be 
brought  back  by  the  addition  of  alkali.  The  medium  used  here  is 
bouillon  containing  an  appropriate  sugar  and  '5  per  cent  of  a  1 
per  cent  watery  solution  of  Grubler's  neutral-red.  In  the  case  of 
the  typhoid  bacillus  no  change  occurs,  but  in  the  case  of  the  b. 
coli  there  is  developed  a  beautiful  canary  yellow  with  a  greenish 

1  A  variety  of  the  organism  which  does  ferment  cane  sugar  has  been 
described  under  the  name  of  the  b.  coli  communior. 


328  TYPHOID  FEVER 

fluorescence.  Fitz- Gerald  and  Dreyer  have  shown  that  an 
important  factor  here  is  the  reaction  of  the  medium,  and  that  the 
effects  of  the  bacteria  may  be  one  of  degree, — under  certain 
circumstances  the  effects  described  as  characteristic  of  the  two 
organisms  may  be  reversed. 

(3)  Formation  of  IndoL — Among  the   bacteria   capable   of 
forming   indol    is   to    be   classed    the    b.    coli.     Indol    can    be 
recognised  in  bouillon  cultures  of  the  b.  coli  three  to  four  days 
old  by  the  usual  tests  (vide  p.  77).     As  there  is  no  evidence 
that  it  can  produce  nitrites  a  small  quantity  of  the  latter  must 
be  added.     The  typhoid  bacillus  never  gives  this  reaction  when 
growing   in    ordinary   conditions,    but    on   the   other   hand,  it 
appears  that  some  varieties  of  the  b.  coli  fail  to  produce  it  also. 
Peckham,   however,  has  found  that  if  the  typhoid  bacillus  be 
grown  in  peptone  solution,  after   a   few   generations  of   three 
days  each  it  may  acquire  the  property  of  producing  indol.     The 
formation  of  indol  by  an  organism  after  the  first  transference  to 
peptone  solution  from  one  of  the  ordinary  media  may,  however, 
be  accepted  as  evidence  in  favour  of  the  organism  not  being  the 
typhoid  bacillus.     It  is  to  be  noted  here  that  the  presence  of 
sugar  in  a  medium  retards  the  production   of  indol  by  the  b. 
coli.     The  indol  reaction  thus  ought  to  be  sought  for  in  a  sugar- 
free  medium. 

(4)  The  Media  of  Capaldi  and  Proskauer. — The  first  of  these 
("No.  1  ")  is  a  medium  free  of  albumin,  in  which  b.  coli  grows 
well  and  freely  produces  acid,  while  the  typhoid  bacillus  hardly 
grows   at   all,    and   certainly   will   produce   no   change  in  the 
reaction.     Its  composition   is  as  follows :    asparagin    '2    parts, 
mannite    *2,    sodium    chloride    "02,    magnesium    sulphate     '01, 
calcium  chloride    '02,    potassium   monophosphate    '2,    distilled 
water  to  100  parts.     The  second  medium  ("No.  2")  contains 
albumin,  and  is  such  that  the  b.  coli  produces  no  acid,  while 
the  typhoid  bacillus  grows  well  and  produces  an  acid  reaction. 
It  consists  of  Witte's  peptone  2  parts,  mannite  *1,  distilled  water 
to  100  parts.     After  the  constituents  of  each  medium  are  mixed 
and  dissolved,  it  is  steamed  for  one  and  a  half  hours  and  then 
made  neutral  to  litmus — the  first  medium,  being  usually  naturally 
acid,  by  sodium  hydrate,  the  second,  being  usually  alkaline,  by 
citric  acid.     The  medium  is  then  filtered,  filled  into  tubes  con- 
taining 5  c.c.,   and  these  are  sterilised.     After  inoculation  for 
twenty  hours  the  reaction  of  the  medium  is  tested  by  adding 
litmus. 

(5)  The  Application  of  the  Agglutination  Test  in  distinguish- 
ing B.  typhosus  from  B.  coli. — The  scope  of  the  application  of 


PATHOLOGICAL   CHANGES  329 

this  test  will  be  discussed  later  (see  Immunity).  Here  we  may 
say  that  a  negative  result  obtained  with  a  suspected  b. 
typhosus  culture  is  of  greater  value  than  a  positive  result 
obtained  with  a  suspected  b.  coli  culture.  The  test  is  to  be 
taken  in  conjunction  with  the  other  means  of  differentiating  the 
two  organisms  (cf.  p.  340). 

It  will  thus  be  seen  that  the  diagnosis  between  the  b. 
typhosus  and  the  b.  coli  is  a  matter  of  no  small  difficulty. 
There  is  no  evidence  that  the  one  organism  ever  passes  into  the 
other.  Great  difficulties  sometimes  arise  in  consequence  of  a 
bacillus  being  found  which,  while  giving  a  number  of  the 
characteristics  of  either  one  or  the  other,  fails  to  give  some  of 
the  characteristic  tests,  or  only  gives  them  very  slowly.  This  is 
especially  true  of  organisms  related  to  the  b.  coli.  It  has 
consequently  become  common  to  speak  of  the  typhoid  group 
and  the  coli  group  in  order  that  such  varieties  may  be  included. 

Pathological  Changes  in  Typhoid  Fever. — Here  we  confine 
our  attention  solely  to  the  bacteriological  aspects  of  the  disease. 
The  inflammation  and  ulceration  in  the  Peyer's  patches  and 
solitary  glands  of  the  intestine  are  the  central  features.  In  the 
early  stage  there  is  produced  an  acute  inflammatory  condition, 
attended  with  extensive  leucocytic  emigration  and  sometimes 
with  small  haemorrhages.  At  this  period  the  typhoid  bacilli  are 
most  numerous  in  the  patches,  groups  being  easily  found  between 
the  cells.  The  subsequent  necrosis  is  evidently  in  chief  part  the 
result  of  the  action  of  the  toxic  products  of  the  bacilli,  which 
gradually  disappear  from  their  former  positions,  though  they 
may  still  be  found  in  the  deeper  tissues  and  at  the  spreading 
margin  of  the  necrosed  area.  They  also  occur  in  the  lymphatic 
spaces  of  the  muscular  coat.  It  is  to  be  remarked  that 
the  number  of  the  ulcers  arising  in  the  course  of  a  case  bears  no 
relation  to  its  .severity.  Small  ulcers  may  occur  in  the  lymphoid 
follicles  of  the  large  intestine. 

The  mesenteric  glands  corresponding  to  the  affected  part  of 
the  intestine  are  usually  enlarged,  sometimes  to  a  very  great 
extent,  the  whole  mesentery  being  filled  with  glandular  masses. 
In  such  glands  there  may  be  acute  inflammation,  and  occasionally 
necrosis  in  patches  occurs.  Sometimes  on  section  the  glands 
are  of  a  pale-yellowish  colour,  the  contents  being  diffluent  and 
consisting  largely  of  leucocytes.  Typhoid  bacilli  may  be  isolated 
both  from  the  glands  and  the  lymphatics  connected  with  them, 
but  the  b.  coli  is  in  addition  often  present. 

The  spleen  is  enlarged, — on  section  usually  of  a  fairly  firm 
consistence,  of  a  reddish-pink  colour,  and  in  a  state  of  congestion. 


330  TYPHOID   FEVER 

Of  all  the  solid  organs  it  usually  contains  the  bacilli  in  greatest 
numbers.  They  can  be  seen  in  sections,  occurring  in  clumps 
between  the  cells,  there  being  no  evidence  of  local  reaction 
round  them  (Fig.  110).  Similar  clumps  may  occur  in  the  liver 
in  any  situation,  and  without  any  local  reaction.  In  this  organ, 
however,  there  are  often  small  foci  of  leucocytic  infiltration,  in 
which,  so  far  as  our  experience  goes,  bacilli  cannot  be  demon- 
strated. The  bacillus  is  found,  often  in  large  numbers,  in  the 
gall-bladder,  where  it  may  persist  for  years.  Clumps  of  bacilli 
may  also  occur  in  the  kidney. 

In  addition  to  these  local  changes  in  the  solid  organs  there  are  also 
widespread  cellular  degenerations  in  the  solid  organs  which  suggest  the 
circulation  of  soluble  poisons  in  the  blood. 

In  the  lungs  there  may  be  bronchitis,  patches  of  congestion  and  of 
acute  broncho-pneumonia.  In  these,  typhoid  bacilli  may  sometimes  be 
observed,  but  evidence  of  a  toxic  action  depressing  the  powers  of  resist- 
ance of  the  lung  tissue  is  found  in  the  fact  that  the  pneumococcus 
frequently  occurs  in  such  complications  of  typhoid  fever. 

The  nervous  system  shows  little  change,  though  meningitis  associated 
either  with  the  typhoid  bacillus,  with  the  b.  coli,  or  with  the  streptococcus 
pyogenes  has  been  observed. 

The  typhoid  bacilli  probably  travel  by  the  blood  stream,  and  they  can 
be  isolated  from  the  blood  much  more  readily  than  was  formerly  supposed. 
Considerable  quantities  of  blood  (say,  4  c.c.)  must  of  course  be  taken 
(v.  p.  68).  They  have  been  found  in  the  roseolar  spots  which  occur  in 
typhoid  fever,  but  it  cannot  be  yet  stated  that  such  spots  are  always  due 
to  the  presence  of  the  bacilli.  The  fact  that  the  typhoid  bacilli  are  usually 
confined  to  certain  organs  and  tissues  shows  that  they  probably  have  a 
selective  action  on  certain  tissues. 

To  sum  up  the  pathology  of  typhoid  fever,  we  have  in  it  a 
disease  the  centre  of  which  lies  in  the  lymphoid  tissue  in  and 
connected  with  the  intestine.  In  this  situation  we  must  have 
an  irritant,  against  which  the  inflammatory  reaction  is  set  up, 
and  which  in  the  intestine  is  sufficiently  powerful  to  cause 
necrosis.  The  affections  of  the  other  organs  of  the  body  suggest 
the  circulation  in  the  blood  of  poisonous  substances  capable  of 
depressing  cellular  vitality,  and  producing  histological  changes. 
The  occurrence  of  bacilli  in  the  blood  and  organs  links  typhoid 
fever  with  septicsemic  processes. 

Suppurations  occurring  in  connection  with  Typhoid  Fever. — 
With  regard  to  the  relation  of  the  typhoid  bacillus  to  such 
conditions,  statements  as  to  its  isolation  from  pus,  etc.,  can  be 
accepted  only  when  all  the  points  available  for  the  diagnosis  of 
the  organism  have  been  attended  to.  On  this  understanding 
the  following  summary  may  be  given  : — In  a  small  proportion 
of  the  cases  examined  the  typhoid  bacillus  has  been  the  only 


PATHOGENIC   EFFECTS   OF   B.   TYPHOSUS     331 

organism  found.  This  has  been  the  case  in  subcutaneous 
abscesses,  in  suppurative  periostitis,  suppuration  in  the  parotid, 
abscesses  in  the  kidneys,  etc.,  and  probably  also  in  one  or  two 
cases  of  ulcerative  endocarditis.  But  in  the  majority  of  cases 
other  organisms,  especially  the  b.  coli  and  the  pyogenic 
micrococci,  have  been  obtained,  the  typhoid  bacillus  having  been 
searched  for  in  vain.  It  has,  moreover,  been  experimentally 
shown,  notably  by  Dmochowski  and  Janowski,  that  suppuration 
can  be  experimentally  produced  by  injection  in  animals,  especially 
in  rabbits,  of  pure  cultures  of  the  typhoid  bacillus,  the  occurrence 
of  suppuration  being  favoured  by  conditions  of  depressed  vitality, 
etc.  These  observers  also  found  that  when  typhoid  bacilli  were 
injected  along  with  pyogenic  staphylococci,  the  former  died  out 
in  the  pus  more  quickly  than  the  latter.  Accordingly,  in  clinical 
cases  where  the  typhoid  bacillus  is  present  alone,  it  is  improbable 
that  other  organisms  were  present  at  an  earlier  date. 

Pathogenic  Effects  produced  in  Animals  by  the  Typhoid 
Bacillus. — There  is  no  disease  known  to  veterinary  science 
which  can  be  said  to  be  identical  with  typhoid,  nor  is  there  any 
evidence  of  the  occurrence  of  the  typhoid  bacillus  under  ordinary 
pathological  conditions  in  the  bodies  of  animals.  Attempts 
to  communicate  the  disease  to  animals  by  feeding  them  on 
typhoid  dejecta  have  been  unsuccessful,  and  though  pathogenic 
effects  have  been  produced  by  introducing  pure  cultures  in 
food,  the  disease  has  usually  borne  no  resemblance  to  human 
typhoid.  The  most  successful  experiments  have  been  those  of 
Remlinger,  who,  by  continuously  feeding  rabbits  on  vegetables 
soaked  in  water  containing  typhoid  bacilli,  produced  in  certain 
cases  symptoms  resembling  those  of  typhoid  fever  (diarrhosa, 
remittent  pyrexia,  etc.).  An  agglutinating  action  was  observed 
in  the  serum,  and  post  'mortem  there  was  congestion  of  the 
Peyerian  patches,  and  typhoid  bacilli  were  isolated  from 
the  spleen. 

While  feeding  experiments  are  thus  rather  unsatisfactory,  the 
same  may  be  said  of  the  results  of  subcutaneous  or  intraperitoneal 
infection.  Here,  again,  pathogenic  effects  can  easily  be  produced 
by  the  typhoid  bacillus,  but  these  effects  are  of  the  nature  of  a 
short  acute  illness  characterised  by  pyrexia,  rapid  loss  of  weight, 
inability  to  take  food,  and  frequently  ending  fatally  in  from 
twenty-four  to  forty-eight  hours.  The  type  of  disease  is  thus  very 
different  from  what  occurs  naturally  in  man.  In  such  injection 
experiments  the  results  vary  considerably,  sometimes  scarcely 
any  effect  being  produced  by  a  large  dose  of  a  culture.  This 
is  no  doubt  due  to  the  fact  that  different  strains  of  the  bacillus 


f*=— — 

,kJbW*-A.W*j7%. 
*F    TMk.  \ 


332  TYPHOID   FEVER 

vary  much  in  virulence.  Ordinary  laboratory  cultures  are  often 
almost  non-pathogenic.  They  can,  however,  be  made  virulent 
in  various  ways.  Sanarelli  used  the  method  of  injecting 
sterilised  cultures  of  the  b.  coli  intraperitoneally  at  the  same 
time  as  the  typhoid  bacillus  was  introduced  subcutaneously. 
After  this  procedure  had  been  repeated  through  a  series  of 
animals  a  culture  of  typhoid  was  obtained  of  exalted  virulence. 
Sidney  Martin  has  obtained  virulent  cultures  by  passing  bacilli, 
derived  directly  from  the  spleen  of  a  person  dead  of  typhoid 
fever,  through  the  peritoneal  cavities  of  a  series  of  guinea-pigs. 

Sanarelli,  studying  the  effects  of  the  intraperitoneal  injection 
of  a  few  drops  of  a  culture  of  highly  exalted  virulence,  found 
that  the  Peyer's  patches  and  solitary  glands  of  the  intestine 
were  enormously  infiltrated,  sometimes  almost  purulent,  and 
that  they  contained  typhoid  bacilli,  as  also  did  the  mesenteric 
lymphatics  and  glands,  and  the  spleen.  These  results  are 
interesting,  but  have  not  been  confirmed. 

The  Toxic  Products  of  the  Typhoid  Bacillus. — Here  very 
little  light  has  been  thrown  on  the  pathology  of  the  disease,  but 
the  general  results  may  be  outlined.  We  may  state  that  there 
exist  in  the  bodies  of  typhoid  bacilli  toxic  substances,  that  in 
artificial  cultures  these  do  not  pass  to  any  great  degree  out  into 
the  surrounding  medium,  and  that  though  they  produce  effects  on 
the  intestine,  there  is  evidence  that  such  effects  are  not  character- 
istic and  not  peculiar  to  the  toxins  of  the  b.  typhosus.  Sidney 
Martin  found  that  the  bodies  of  bacteria  killed  by  chloro- 
form vapour  were  very  toxic, — more  so  than  filtered  cultures. 
Diarrhoea  was  a  constant  symptom  after  injection,  but  no  change 
in  the  Peyerian  patches  was  observed.  Martin  found  that 
virulent  cultures  of  the  b.  coli  gave  similar  results  when 
similarly  treated.  Allan  Macfadyen,  by  grinding  up  typhoid 
bacilli  frozen  solid  by  liquid  air,  produced  a  fluid  whose  toxic 
effect  he  attributed  to  the  presence  of  the  intracellular  poisons. 

The  Immunisation  of  Animals  against  the  Typhoid 
Bacillus. — Earlier  observers  had  been  successful  in  accustoming 
mice  to  the  typhoid  bacillus  by  the  successive  injections  of  small 
and  gradually  increasing  doses  of  living  cultures  of  the  bacillus. 
Later,  Brieger,  Kitasato,  and  Wassermann  found  that  the  bacillus 
when  modified  by  being  grown  in  a  bouillon  made  from  an 
extract  of  the  thymus  gland  no  longer  killed  mice  and  guinea- 
pigs  These  animals  after  injection  were  moreover  immune,  and 
it  was  also  found  that  the  serum  of  a  guinea-pig  thus  immunised 
could,  if  transferred  to  another  guinea-pig,  protect  the  latter 
from  the  subsequent  injection  of  a  dose  of  typhoid  bacilli  to 


PATHOGENICITY   OF   B.    COLI  333 

which  it  would  naturally  succumb.  Chantemesse  and  Widal, 
Sanarelli,  and  also  Pfeiffer,  succeeded  in  immunising  guinea-pigs 
against  the  subsequent  intraperitoneal  injection  of  virulent 
living  typhoid  bacilli,  by  repeated  and  gradually  increasing 
intraperitoneal  or  subcutaneous  doses  of  dead  typhoid  cultures 
in  bouillon.  Experiments  performed  with  serum  derived  from 
typhoid  patients  and  convalescents  indicate  that  similar  effects 
occur  in  those  who  have  successfully  resisted  the  natural  disease. 
The  serum  of  such  patients  has  antibacterial  powers,  but 
there  is  no  evidence  that  it  contains  any  antitoxic  bodies  (see 
chapter  on  Immunity).  Pfeiffer,  for  example,  found  on  adding 
serum  from  typhoid  convalescents  to  the  bodies  of  typhoid 
bacilli  killed  by  heat,  and  injecting  the  mixture  into  guinea- 
pigs,  that  death  took  place  as  in  control  animals  which  had 
received  these  toxic  agents  alone.  Pfeiffer  also  found  that  by 
using  the  serum  of  immunised  goats,  he  could,  to  a  certain 
extent,  protect  other  animals  against  the  subsequent  injection 
of  virulent  living  typhoid  bacilli.  On  trying  to  use  the  agent 
in  a  curative  way,  i.e.  injecting  it  only  after  the  bacilli  had 
begun  to  produce  their  effects,  he  got  little  or  no  result. 

The  Pathogenicity  of  the  B.  coli  and  its  Relation  to  that  of  the 
Typhoid  Bacillus. — We  have  already  seen  that  the  b.  coli  is  probably 
responsible  for  the  occurrence  of  some  of  the  abscesses  which  follow 
typhoid  fever.  It  is  also  apparently  the  cause  of  some  cases  of  summer 
diarrhoea  (cholera  nostras),  of  infantile  diarrhcea,  and  of  some  food 
poisonings.  Its  numbers  in  the  intestine  are  greatly  increased  during 
typhoid  fever,  and  also  during  any  pathological  condition  affecting  the 
intestine.  Intraperitoneal  injection  in  guinea-pigs  is  often  fatal.  Sub- 
cutaneous injection  may  result  in  local  abscesses,  and  sometimes  in  death 
from  cachexia.  Sanarelli  found  that  the  b.  coli  isolated  from  typhoid 
stools  was  much  more  virulent  than  when  isolated  from  the  stools  of 
healthy  persons.  He  holds  that  the  increase  in  virulence  is  due  to  the 
effect  of  typhoid  toxins.  This  increased  virulence  of  the  b.  coli  in  the 
typhoid  intestine  makes  it  possible  that  some  of  the  pathological  changes 
in  typhoid  may  be  due,  not  to  the  typhoid  bacillus,  but  to  the  b.  coli. 
Some  of  the  general  symptoms  may  be  intensified  by  the  absorption  of 
toxic  products  formed  by  it  and  by  other  organisms.  It  is  to  be  noted 
that  lesions  produced  in  guinea-pigs  are  very  similar  to  those  of  the  b. 
typhosus.  Differences  of  behaviour  of  the  two  bacilli  in  connection  with 
their  pathological  effects  have  been  brought  forward  as  confirmatory  of 
the  fact  of  their  being  distinct  species.  Thus  Sanarelli  accustomed  the 
intestinal  mucous  membrane  of  guinea-pigs  to  toxins  derived  from  an  old 
culture  of  the  b.  coli,  by  introducing  day  by  day  small  quantities  of  the 
latter  into  the  stomach.  When  a  relatively  large  dose  could  be  tolerated, 
it  was  found  that  the  introduction  in  the  same  way  of  a  small  quantity 
of  typhoid  toxin  was  still  followed  by  fatal  result.  Pfeiffer  also  found 
that  while  the  serum  of  convalescents  from  typhoid  paralysed  the 
typhoid  bacilli,  it  had  no  more  effect  on  similar  numbers  of  b.  coli  than 
the  serum  of  healthy  men. 


334  TYPHOID   FEVER 

General  View  of  the  Relationship  of  the  B.  typhosus  to 
Typhoid  Fever. — 1.  We  have  in  typhoid  fever  a  disease  having 
its  centre  in  and  about  the  intestine,  and  acting  secondarily  on 
many  other  parts  of  the  body.  In  the  parts  most  affected  there 
is  always  a  bacillus  present,  microscopically  resembling  other 
bacilli,  especially  the  b.  coli  which  is  a  normal  inhabitant  of 
the  animal  intestine.  This  bacillus  can  be  isolated  from  the 
characteristic  lesions  of  the  disease  and  from  other  parts  of  the 
body  as  described,  and  further,  it  is  found  by  culture  and  serum 
reactions  to  differ  from  other  organisms.  Here  the  important 
point  is  that  a  bacillus  giving  all  the  reactions  of  the  typhoid 
bacillus  has  never  been  isolated  except  from  cases  of  typhoid 
fever,  or  under  circumstances  that  make  it  possible  for  the 
bacillus  in  question  to  have  been  derived  from  a  case  of  typhoid 
fever. 

2.  A  difficulty  in  the  way  of  accepting  the  etiological  relation- 
ship of  the  b.  typhosus  lies  in  the  comparative  failure  of  attempts 
to  cause  the  disease  in  animals.     We  have  noted,  however,  that 
in  nature  animals  do  not  suffer  from  typhoid  fever. 

3.  The  observations  of  Pfeiffer  and  others  on  the  protective 
power  against  typhoid  bacilli  shown,  on  testing  in  animals,  to 
belong  to  the  serum  of  typhoid  patients  and  convalescents,  and 
the  peculiar  action  of  such  serum  in  immobilising  and  causing 
clumping  of  the  bacilli  (vide  infra)  are  also  of  great  importance 
as  indicating  an  etiological  relationship  between  the  bacillus  and 
the  disease.     Additional  important  evidence  is  found  in  the  fact 
that  vaccination  by  means  of  the  dead  bacilli  (vide  infra)  has  a 
marked  effect  in  preventing  the  disease  from  arising  in  a  popula- 
tion exposed  to  infection,  and  also  in  lowering  the  mortality 
when  the  fever  attacks  those  who  have  been  inoculated.     These 
facts  may  thus  be  accepted  as  indirect  but  practically  conclusive 
evidence  of  the  pathogenic  relationships  of  the  typhoid  bacillus 
to  the  disease. 

According  to  our  present  results  we  must  thus  hold  that  the 
b.  typhosus  constitutes  a  distinct  species  of  bacterium,  and 
that  it  is  the  cause  of  typhoid  fever.  Evidence  of  an  important 
nature  confirmatory  of  this  view  is,  we  think,  found  in  the  fact 
that  cases  have  occurred  where  bacteriologists  have  accidentally 
infected  themselves  by  the  mouth  with  pure  cultures  of  the 
typhoid  bacillus,  and  after  the  usual  incubation  period  has 
developed  typhoid  fever.  Several  cases  of  this  kind  have  been 
brought  to  our  notice  and  are  not,  we  think,  vitiated  by  the  fact 
that  other  similar  instances  have  occurred  without  the  subsequent 
development  of  illness.  These  latter  would  be  accounted  for  by 


PARATYPHOID   BACILLUS  335 

a  low  degree  of  susceptibility  on  the  part  of  the  individual  or  to 
a  want  of  pathogenicity  in  the  cultures. 

The  Paratyphoid  Bacillus. — This  organism,  which  was  when 
first  described  often  denominated  the  paracolon  bacillus,  was 
primarily  isolated  from  abscesses  occurring  in  apparently  non- 
typhoid  cases.  Widal  noted  its  resemblances  to  b.  typhosus  and 
b.  coli,  from  the  latter  of  which  it  differed  in  not  producing 
indol  and  in  not  fermenting  lactose.  Gwynn  first  isolated  it 
from  the  blood  of  a  case  presenting  typhoid  symptoms,  and  since 
then  it  has  been  recognised  as  being  the  probable  cause  of  the 
disease  effects  in  about  3  per  cent  of  cases  which  clinically  are  to 
be  described  as  typhoid  fever.  The  case-mortality  in  paratyphoid 
fever  is  low,  being  only  from  1  to  2  per  cent.  The  organism 
has  been  isolated  from  the  blood,  the  roseolar  spots,  and  from 
the  stools, — plates  of  MacConkey's  bile -salt  neutral -red  agar 
with  1  per  cent  lactose  added  being  recommended  for  use  here. 
Several  strains  showing  slight  differences  in  culture  reactions 
have  been  obtained.  Generally  speaking,  the  cultural  reactions 
resemble  those  of  b.  coli,  though  the  growth  on  potato  some- 
times presents  typhoid  characteristics.  It  produces  no  indo], 
or  at  least  (with  some  strains)  merely  a  trace,  and  its  action 
on  sugars  also  differs.  With  regard  to  the  latter,  different 
results  have  been  obtained  by  different  observers,  but  there  is 
general  agreement  that,  like  the  b.  coli,  it  produces  acid  and  gas 
in  glucose,  l^vulose,  sorbite,  mannite,  dextrin,  maltose,  dulcite, 
galactose,  and  arabinose;  but  in  lactose,  like  typhoid,  it  either 
originates  no  change  or  only  slight  acid  production  without  any 
gas  formation.  It  also  causes  no  change  in  cane  sugar,  erythrite, 
salicin,  inulin,  and  raffinose.  Probably  the  most  important 
reactions  which  will  in  any  case  aid  in  the  recognition  of  the 
paratyphoid  bacillus  are  the  agglutinating  reactions.  The 
serum  of  a  paratyphoid  patient  will  agglutinate  the  bacillus  in 
high  dilutions.  Observations  on  the  behaviour  of  such  sera 
towards  the  b.  typhosus  have  in  different  cases  yielded  some 
discordant  results,  but  to  speak  generally,  it  may  be  said  that 
usually  a  very  much  stronger  concentration  is  necessary  to  give 
clumping,  and  often  a  paratyphoid  serum  will  not  clump  the 
typhoid  bacillus  except  in  such  concentrations  as  might  give 
similar  effects  when  normal  sera  are  under  observation.  When 
any  serum  clumps  both  the  paratyphoid  and  the  typhoid  bacilli 
the  more  closely  the  maximal  clumping  dilutions  correspond,  the 
more  likely  is  the  case  to  be  typhoid  fever ;  on  the  other  hand, 
if  a  high  dilution  will  clump  the  paratyphoid  bacillus,  while 
a  low  dilution  is  necessary  for  the  typhoid  bacillus,  then 


336  TYPHOID   FEVER 

the  case  is  likely  to  be  paratyphoid  fever.  With  regard  to  the 
effects  of  other  sera  on  the  paratyphoid  bacillus,  it  may  be  said 
that  usually  a  typhoid  serum  will  require  to  be  used  in  greater 
concentrations  to  clump  this  bacillus  than  are  necessary  to  obtain 
an  effect  with  the  typhoid  bacillus  itself.  Similar  effects  are 
observed  when  the  sera  of  animals  immunised  against  Gaertner's 
bacillus  or  the  bacillus  of  psittacosis  are  used.  In  all  serum 
tests  the  essential  point  is  that  deductions  should  alone  be  based 
on  comparative  observations  of  the  highest  dilutions  in  which 
a  clumping  effect  is  produced  with  any  series  of  organisms 
compared. 

As  has  been  indicated,  a  disease  resembling  typhoid  fever  is 
not  the  only  condition  originated  by  the  paratyphoid  bacillus. 
The  organism  has  been  isolated  from  cases  of  bone  abscess,  from 
orchitis,  and  in  Widal's  case  from  a  thyroid  abscess,  and  in  such 
cases  the  history  of  a  previous  typhoid-like  illness  may  not  be 
elicited.  It  has  also  been  found  in  ordinary  faeces.  In  animal 
experiments  it  produces  in  rabbits  and  guinea-pigs  a  fatal  ill- 
ness of  a  septicsemic  type  with  serous  inflammations. 

Bacillus  Enteritidis  (Gaertner).—  In  1888  Gaertner,  in 
investigating  a  number  of  cases  of  illness  resulting  from  eating 
the  flesh  of  a  diseased  cow,  isolated,  from  the  meat  and  from  the 
spleen  of  a  man  who  died,  a  bacillus  closely  resembling  the 
typhoid  bacillus.  Since  then  a  great  number  of  outbreaks  of 
gastro-enteritis  due  to  eating  diseased  meat  have  been  inquired 
into,  and  very  frequently  similar  bacilli  have  been  found  both  in 
the  stools  and  in  the  organs.  These  bacilli  closely  resemble  the 
paratyphoid  organism, — indol  is  not  produced,  and  generally 
speaking  the  fermentations  of  sugars  also  correspond.  With 
regard  to  the  latter  it  may,  however,  be  said  that,  according  to 
some,  lactose  is  fermented,  while  other  observers  have  found  this 
not  to  be  the  case.  No  doubt  different  strains  differ  somewhat 
from  one  another.  Here  again  much  information  may  be 
obtained  from  the  agglutinating  properties  of  the  serum  and  also 
from  the  effects  on  suspicious  bacilli  of  the  sera  of  animals 
immunised  against  other  strains  and  other  members  of  the  coli 
group.  It  has  also  been  found  that  the  serum  of  persons  suffer- 
ing from  meat  poisoning  sometimes  clumps  the  typhoid  bacillus, 
though  a  higher  concentration  is  required  than  in  the  case  of 
Gaertner's  bacillus.  The  Gaertner  group  of  organisms  is  very 
pathogenic  for  laboratory  animals.  Often,  whatever  the  channel 
of  infection,  there  is  intense  haemorrhagic  enteritis,  and  very 
usually  there  is  a  septicaemia  with  the  occurrence  of  serous  inflam- 
mations ;  the  bacilli  are  recoverable  from  the  solid  organs  and 


PSITTACOSIS   BACILLUS  337 

often  from  the  blood.  In  man,  as  the  name  of  the  bacillus 
indicates,  the  symptoms  are  centred  in  the  intestine,  where  there 
is  usually  marked  inflammation  of  the  mucous  membrane,  some- 
times attended  with  haemorrhage  into  it ;  evidence  of  a  septicaemic 
condition  may  also  exist.  Infection  may  take  place  by  the 
bacillus  itself  where  meat  has  been  insufficiently  cooked  or 
merely  pickled,  and  here  the  illness  usually  appears  within 
twenty-four  hours  of  the  food  being  partaken  of,  but  symptoms 
may  appear  almost  at  once,  in  which  case  they  are  no  doubt  due 
to  the  action  of  toxins ;  here  it  is  important  to  note  that  the 
poisons  formed  by  this  group  of  organisms  are  relatively  heat- 
resisting,  so  that  boiling  for  a  time  does  not  destroy  the  toxicity. 
In  cases  of  Gaertner  bacillus  poisoning,  the  animal  whose 
carcase  is  suspected  has  usually  been  found  to  have  been  itself 
suffering  from  the  action  of  the  bacillus,  but  cases  of  meat 
poisoning  also  occur  where  the  meat  of  a  healthy  animal  becomes 
infected  subsequently  to  slaughter  with  organisms  pathogenic  to 
man.  In  such  cases  these  organisms  are  often  varieties  of  the 
b.  coli  group,  and  indeed  the  b.  coli  itself  may  be  the  cause  of 
meat  poisoning. 

The  Psittacosis  Bacillus.— When  parrots  are  imported  from  the 
tropics  in  large  numbers  many  die  of  a  septicsemie  condition  in  which  an 
enteritis,  it  may  be  hsemorrhagic,  is  a  marked  feature.  There  is  intense 
congestion  of  all  the  organs  and  peritoneal  ecchymoses.  From  the 
spleen,  bone  marrow,  and  blood  there  has  been  isolated  a  short, 
actively,  motile  bacillus  with  rounded  ends  which  does  not  stain  by 
Gram's  method.  It  groivs  on  all  ordinary  media,  and  on  potato  resembles 
b.  coli.  It  does  not  liquefy  gelatin,  does  not  ferment  lactose,  does  not 
curdle  milk,  and  gives  no  indol  reaction.  Culturally  the  organism  is 
practically  indistinguishable  from  the  two  bacilli  last  described.  The 
parrot  is  most  susceptible  to  its  action,  but  it  also  causes  a  fatal 
hsemorrhagic  septicaemia  in  guinea-pigs,  rabbits,  mice,  pigeons,  and 
fowls,  the  bacilli  after  death  being  chiefly  in  the  solid  organs.  From 
affected  parrots  the  disease  appears  to  be  readily  communicable  to  man, 
chiefly,  it  is  probable,  from  the  feathers  being  soiled  by  infective 
excrement.  Several  small  epidemics  have  been  recognised  and  in- 
vestigated in  Paris.  After  about  ten  days'  incubation,  headache, 
fever,  and  anorexia  occur,  followed  by  great  restlessness,  delirium,  vomit- 
ing, often  diarrhoea,  and  albuminuria.  Frequently  broncho-pneumonia 
supervenes,  and  a  fatal  result  has  followed  in  about  a  third  of  the  cases 
observed.  The  organism  has  been  isolated  from  the  blood  of  the  heart. 
The  psittacosis  bacillus  is  evidently  one  of  the  typhoid  group,  a  fact 
which  is  further  borne  out  by  the  observation  that  it  is  clumped  by  a 
typhoid  serum — 1  :  10  (normal  serum  having  no  result).  The  clumping 
is,  however,  said  to  be  incomplete,  as  the  bacilli  between  the  clumps  may 
retain  their  motility.  It  differs  from  the  typhoid  bacillus  in  its  growth 
on  potatoes  and  in  its  pathogenicity. 

The  Serum  Diagnosis  of  Typhoid  Fever. — This  method  of 

22 


338  TYPHOID   FEVER 

diagnosis  is  based  on  the  fact  that  living  and  actively  motile 
typhoid  bacilli,  if  placed  in  the  diluted  serum  of  a  patient  suffer- 
ing from  typhoid  fever,  within  a  very  short  time  lose  their 
motility  and  become  aggregated  into  clumps.  We  shall  find 
(see  Immunity)  that  in  many  diseases  the  serum  has  this  property 
of  causing  agglutination  of  cultures  of  the  causal  bacterium. 
The  principles  on  which  the  possession  of  the  faculty  depends  and 
also  its  significance,  are  obscure,  and  in  the  case  of  the  typhoid 
bacillus  we  do  not  know  the  true  interpretation  of  some  of  the 
facts  which  have  been  observed. 

The  methods  by  which  the  test  can  be  applied  have  already 
been  described  (p.  109). 

(1)  It  will  be  there  seen  that  the  loss  of  motility  and  clumping 
may  be  observed  microscopically.     If  a  preparation  be  made  by 
the  method  detailed  (typhoid  serum  in  a  dilution  of,  say,  1  :  30 
having  been  employed),  and  examined  at  once  under  the  micro- 
scope, the  bacilli  will  usually  be  found  actively  motile,  darting 
about  in  all  directions.     In  a  short  time,  however,  these  move- 
ments gradually  become  slower,  the  bacilli  begin  to  adhere  to  one 
another,  and  ultimately  become  completely  immobile  and  form 
clumps  by    their  aggregation,   so  that  no  longer  are  any  free 
bacilli   noticeable   in   the  preparation.     When  this  occurs  the 
reaction  is  said  to  be  complete.     If  the  clumps  be  watched  still 
longer   a  swelling  up  of  the  bacilli  will  be   observed,  with  a 
granulation  of  the  protoplasm,   so   that  their  forms  can   with 
difficulty  be  recognised.     In  a  preparation  similarly  made  with 
non-typhoid  serum  the  individual  bacilli  can  be  observed  separate 
.and  actively  motile  for  many  hours. 

(2)  A  corresponding  reaction    visible    to   the   naked   eye   is 
obtained  by  the  "  sedimentation  test,"  the  method  of  applying 
which  has  also  been  described  (p.   111).     Here  at  the  end  of 
twenty-four  hours  the  bacilli  form  a  mass  like  a  precipitate  at 
the   bottom  of  the  mixture  of  bacterial  emulsion  and    diluted 
typhoid  serum,  while  the  upper  part  remains  clear.     A  similar 
preparation  made  with  normal  serum  shows  a  diffuse  turbidity 
at  the  end  of  twenty-four  hours.     The  test  in  this  form  has  the 
disadvantage  of  taking  longer  time  than  the  microscopic  method, 
but  it  is  useful  as  a  control ;  in  nature  it  is  similar. 

Such  is  what  occurs,  in  the  case  of  a  typical  reaction.  The 
value  of  the  method  as  a  means  of  diagnosis  largely  depends  on 
attention  to  several  details.  The  race  of  typhoid  bacillus 
employed  is  important.  All  races  do  not  give  uniformly  the 
same  results,  though  it  is  not  known  on  what  this  difference  of 
susceptibility  depends.  A  race  must  therefore  be  selected 


SERUM   DIAGNOSIS  339 

which  gives  the  best  result  in  the  greatest  number  of  undoubted 
cases  of  typhoid  fever,  and  which  gives  as  little  reaction  as 
possible  with  normal  sera  or  sera  derived  from  other  diseases. 
This  latter  point  is  important,  as  some  races  react  very  readily  to 
non-typhoid  sera.  Again,  care  must  be  taken  as  to  the  state  of 
the  culture  used.  The  suitability  of  a  culture  may  be  impaired 
by  varying  the  conditions  of  its  growth.  Continued  growth  of  a 
race  at  37°  C.  makes  it  less  suitable  for  use  in  the  test,  as  the 
bacilli  tend  naturally  to  adhere  in  clumps,  which  may  be 
mistaken  for  those  produced  by  the  reaction.  Wyatt  Johnson 
recommended  that  the  stock  culture  should  be  kept  growing  on 
agar  at  room  temperature  and  maintained  by  agar  sub-cultures 
made  once  a  month.  For  use  in  applying  the  test,  bouillon 
sub-cultures  are  made  and  incubated  for  twenty-four  hours  at 
37°  C.  The  relation  of  the  dilution  of  the  serum  to  the 
occurrence  of  clumping  is  most  important.  It  has  been  found 
that  if  the  degree  of  dilution  be  too  small  a  non-typhoid  serum 
may  cause  clumping.  If  possible,  observations  should  always  be 
made  with  dilutions  of  1  :  10,  1  :  30,  1  :  60,  1  :  100.  To  speak 
generally,  the  more  dilute  the  serum  the  longer  time  is  necessary 
for  a  complete  reaction.  Some  typhoid  sera  have,  however, 
very  powerful  agglutinating  properties,  and  may  in  a  compara- 
tively short  time  produce  a  reaction  when  diluted  many  hundreds 
of  times.  With  a  too  dilute  serum  not  only  may  the  reaction 
be  delayed,  but  it  may  be  incomplete, — the  clumps  formed  being- 
small  and  many  bacilli  being  left  free.  These  latter  may  either 
have  been  rendered  motionless  or  they  may  still  be  motile.  No 
diagnosis  is  conclusive  which  is  founded  on  the  occurrence  of 
such  an  incomplete  clumping  alone.  Seeing  that  low  dilutions 
sometimes  give  a  reaction  with  non-typhoid  sera,  it  is  important 
to  know  what  is  the  highest  dilution  at  which  complete 
clumping  indicates  a  positive  reaction.  The  general  consensus 
of  opinion,  with  which  our  own  experience  agrees,  is  that  when 
a  serum  in  a  dilution  of  1  :  30  causes  complete  clumping  in  half 
an  hour,  it  may  safely  be  said  that  it  has  been  derived  from  a 
case  of  typhoid  fever.  Suspicion  should  be  entertained  as  to 
the  diagnosis  if  a  lower  dilution  is  required,  or  if  a  longer  time 
is  required. 

The  reaction  given  by  the  serum  in  typhoid  fever  usually 
begins  to  be  observed  about  the  seventh  day  of  the  disease, 
though  occasionally  it  has  been  found  as  early  as  the  fifth  clay, 
and  sometimes  it  does  not  appear  till  the  third  week  or  later. 
Usually  it  becomes  gradually  more  marked  as  the  disease 
advances,  and  it  is  still  given  by  the  blood  of  convalescents  from 


340  TYPHOID   FEVER 

typhoid,  but  cases  occur  in  which  it  may  permanently  disappear 
before  convalescence  sets  in.  How  long  it  lasts  after  the  end  of 
the  disease  has  not  yet  been  fully  determined,  but  in  many  cases 
it  has  been  found  after  several  months  or  longer.  As  a  rule,  up 
to  a  certain  point,  the  reaction  is  more  marked  where  the  fever 
is  of  a  pronounced  character,  whilst  in  the  milder  cases  it  is  less 
pronounced.  In  certain  grave  cases,  however,  the  reaction  has 
been  found  to  be  feeble  or  almost  absent,  and  accordingly  some 
hold  that  a  feeble  reaction  when  the  disease  is  manifestly  severe 
is  of  bad  omen.  In  some  cases  which  from  the  clinical  symptoms 
were  almost  certainly  typhoid,  the  reaction  has  apparently  been 
found  to  be  absent.  Such  cases  should  always  be  investigated 
from  the  point  of  view  of  their  possibly  being  paratyphoid  fever. 

It  has  been  found  that  the  reaction  is  not  only  obtained  with 
living  bacilli,  but  in  certain  circumstances  also  with  bacilli 
that  have  been  killed  by  heating  at  60°  C.  for  an  hour, — if 
a  higher  temperature  be  used,  sensitiveness  to  agglutination  is 
impaired.  The  capacity  is  also  still  retained  if  a  germicide  be 
employed.  Here  Widal  recommends  the  addition  of  one  drop  of 
formalin  to  150  drops  of  culture.  The  reaction,  however,  tends 
to  be  less  complete. 

Besides  the  blood  serum  it  has  been  found  that  the  reaction 
is  given  in  cases  of  typhoid  fever  by  pericardial  and  pleural 
effusions,  by  the  bile  and  by  the  milk,  and  also  to  a  slight 
degree  by  the  urine.  The  blood  of  a  foetus  may  have  little 
agglutinating  effect  though  that  of  its  mother  may  have  given 
a  well-marked  reaction ;  sometimes,  however,  the  foetal  blood 
gives  a  well-marked  reaction.  It  may  here  also  be  mentioned 
that  a  serum  will  stand  exposure  for  an  hour  at  58°  C.  without 
having  its  agglutinating  power  much  diminished.  Higher  tem- 
peratures, however,  cause  the  property  to  be  lost. 

The  Agglutination  of  Organisms  other  than  the  B.  Typhosus 
by  Typhoid  Serum. — It  was  at  first  thought  that  the  reaction  in 
typhoid  fever  would  afford  a  reliable  method  of  distinguishing 
the  typhoid  bacillus  from  the  b.  coli.  Though  many  races  of 
the  latter  give  no  reaction  with  a  typhoid  serum,  there  are  others 
which  react  positively.  Usually,  however,  a  lower  dilution  and 
a  longer  time  are  required  for  a  result  to  be  obtained,  and  the 
reaction  is  often  incomplete.  It  has  also  been  found  that  other 
organisms  belonging  to  the  typhoid  group  (v.  p.  335)  react  in  a 
similar  way.  The  reaction  as  a  method  of  distinguishing  between 
these  forms  is  thus  not  absolutely  reliable,  but  in  certain  cases  it 
is  of  great  value  in  giving  confirmation  to  other  tests.  The  im- 
portant point  here  is  the  determination  of  the  highest  dilution 


SERUM   DIAGNOSIS  341 

with  which  clumping  is  obtained.  There  is  a  point  in  this 
connection  regarding  which  further  light  is  required.  Many 
races  of  b.  coli  in  use  have  been  isolated  from  typhoid  cases, 
and  we  as  yet  do  not  know  what  effect  a  sojourn  in  such  circum- 
stances may  have  on  its  subsequent  sensitiveness  to  agglutination 
by  typhoid  serum.  Again,  Christophers  has  pointed  out  that  a 
large  proportion  of  sera  from  normal  persons  or  from  those 
suffering  from  diseases  other  than  typhoid  will  clump  the  b.  coli 
in  dilutions  of  from  1  :  20  to  1  :  200,  and  no  doubt  many  of  the 
reactions  shown  by  typhoid  sera  towards  b.  coli  are  due  to  the 
pre-existence  in  the  individuals  of  an  agglutinative  property 
towards  the  latter  bacillus. 

With  regard  to  the  value  of  the  serum  reaction  there  is  little 
doubt.  In  nearly  95  per  cent  of  cases  of  typhoid  it  can  be 
obtained  in  such  a  form  that  no  difficulty  is  experienced  if  the 
precautions  detailed  above  are  observed.  The  causes  of  possible 
error  may  be  summarised  as  follows  :  the  serum  of  the  person 
may  naturally  have  the  capacity  of  clumping  typhoid  bacilli ; 
there  may  have  been  an  attack  of  typhoid  fever  previously  with 
persistence  of  agglutinative  capacity ;  the  case  may  be  one  of 
disease  caused  by  an  allied  bacillus ;  the  disease  may  have  a 
quite  different  cause,  and  yet  the  serum  may  react  with  typhoid 
bacilli ;  the  disease  may  be  typhoid  fever  and  yet  no  reaction 
may  occur.  The  most  important  of  these  sources  of  error  is 
that  .with  which  diseases  caused  by  allied  organisms  are 
concerned,  as  it  is  probable  that  all  the  forms  which  these  take 
in  men  have  not  been  recognised.  The  very  wide  application 
of  the  reaction  has  elicited  the  fact  that  it  is  given  in  many 
cases  of  slight,  transient,  and  ill -denned  febriculse,  which  occur 
especially  when  typhoid  fever  is  prevalent.  Some  of  these  may 
be  aborted  typhoid,  some  may  be  paratyphoid.  There  is  no  doubt 
that,  if  all  the  facts  are  taken  into  account,  the  cases  where  the 
reaction  gives  undoubtedly  correct  information  so  far  outnumber 
those  in  which  an  error  may  be  made  that  it  must  be  looked  on 
as  a  most  valuable  aid  to  diagnosis.  In  conclusion  here  we  may 
say  that  the  fact  of  a  typhoid  serum  clumping  allied  bacilli  in 
no  way,  so  far  as  our  present  knowledge  goes,  justifies  doubt 
being  cast  on  the  specific  relation  of  the  typhoid  bacillus  to 
typhoid  fever. 

In  connection  with  the  phenomenon  that  a  serum  either  from 
a  normal  person  or  a  typhoid  patient  may  clump  several  varieties 
of  bacteria,  some  points  arise.  The  theoretical  consideration  of 
agglutination  is  reserved  for  the  chapter  on  Immunity,  but  here 
it  may  be  said  that  agglutinating  properties  may  be  present 


342  TYPHOID   FEVER 

normally  in  a  serum  or  they  may  be  originated  by  an  animal 
being  infected  with  a  particular  bacterium.  As  the  result  of 
injecting  a  bacterium  not  only  may  agglutinins  capable  of 
acting  on  that  bacterium  appear  in  the  serum,  but  the  serum  may 
become  capable  of  agglutinating  other,  and  especially  kindred, 
bacteria ;  further,  any  normal  agglutinins  for  the  infecting 
bacterium  present  in  the  serum  may  be  increased  in  amount. 
The  agglutinin  acting  on  the  infecting  organism  has  been  called 
the  primary  or  homologous  agglutinin,  while  the  others  have  been 
called  the  secondary  or  heterologous  agglutinins.  But  besides 
what  we  know  to  be  a  fact,  that  infection  by  a  single  bacillary 
species  can  originate  agglutinins  acting  both  on  itself  and  on  allied 
species,  we  must  consider  the  possibility  of  infections  by  more 
than  one  species  occurring  in  an  animal,  e.g.  b.  typhosus  with  b. 
coli  or  with  b.  paratyphosus.  In  such  a  case  each  organism 
may  originate  its  primary  agglutinin  so  that  the  presence  of 
multiple  agglutinins  in  a  serum  may  really  be  an  indication  of 
a  mixed  infection.  Some  attention  has  been  directed  to  the 
diagnosis  and  differentiation  of  these  conditions.  Castellani 
has  introduced  a  method  for  their  investigation.  This  depends 
on  the  capacity  manifested  by  bacteria  of  absorbing  the  ag- 
glutinins from  a  serum.  A  small  quantity  of  the  agglutinating 
serum,  say  '5  c.c.,  is  taken  either  pure  or  diluted  with  bouillon, 
there  are  added  4  to  8  loops  of  an  agar  culture  of  the  germ  which 
originated  it,  the  mixture  is  well  shaken  and  set  at  37°  C.  for  12 
hours.  Clumping  of  course  occurs,  and  the  clumps  fall  to  the 
bottom  of  the  tube.  The  supernatant  fluid  is  pipetted  off  and 
is  available  for  further  tests.  Castellani  studied  the  primary  and 
secondary  agglutinins  produced  in  infections  in  rabbits;  he  found 
that  when  an  animal  had  been  infected  with  b.  typhosus  this 
organism  would  absorb  from  its  serum  not  only  the  primary 
typhoid  agglutinins  but  also  such  secondary  agglutinins  as  those 
acting  on  the  b.  coli.  If,  however,  an  animal  had  undergone 
infection  with,  say,  both  the  b.  typhosus  and  the  b.  coli,  then  the 
b.  typhosus  could  not  absorb  from  its  serum  the  b.  coli  (primary) 
agglutinin.  Castellani  thus  put  forward  the  view  that  by  this 
means  primary  could  be  differentiated  from  secondary  agglutinins, 
and  therefore  pure  could  be  differentiated  from  mixed  infections. 
There  is  little  doubt  that  this  view  possesses  considerable  validity, 
though  it  is  probably  not  of  universal  applicability.  Safe 
deductions  can  only  be  drawn  when  any  serum  is  tested  with 
several  species  of  fairly  closely  related  organisms,  such  as  those 
of  the  coli  group.  Especially  is  it  necessary  that  the  highest 
dilutions  in  which  agglutination  occurs  should  be  compared.  If 


VACCINATION   AGAINST   TYPHOID  343 

such  precautions  be  adopted  the  absorption  method  can  be  util- 
ised for  the  differentiation  of  the  typhoid  and  paratyphoid  organ- 
isms and  their  infections  and  for  similar  investigations. 

Vaccination  against  Typhoid. — The  principles  of  the  im- 
munisation of  animals  against  typhoid  bacilli  have  been  applied 
by  Wright  and  Semple  to  man  in  the  following  way.  Typhoid 
bacilli  are  obtained  of  such  virulence  that  a  quarter  of  a  twenty- 
four  hours'  old  sloped  agar  culture  when  administered  hypo- 
dermically  will  kill  a  guinea-pig  of  from  350  to  400  grammes. 
Vaccination  can  be  accomplished  by  such  a  culture  emulsified 
in  bouillon,  and  killed  by  heating  for  five  minutes  at  60°  C. 
For  use,  from  one-twentieth  to  one-fourth  of  the  dead  culture  is 
injected  hypodermically,  usually  in  the  flank.  The  vaccine  now 
used,  however,  actually  consists  of  a  portion  of  a  bouillon 
culture  similarly  treated.  The  effects  of  the  injection  are  some 
tenderness  locally  and  in  the  adjacent  lymphatic  glands,  and  it 
may  be  local  swelling,  all  of  which  come  on  in  a  few  hours,  and 
may  be  accompanied  by  a  general  feeling  of  restlessness  and  a 
rise  of  temperature,  but  the  illness  is  over  in  twenty-four  hours. 
During  the  next  ten  days  the  blood  of  the  individual  begins  to 
manifest,  when  tested,  an  agglutination  reaction,  and  further, 
Wright  has  found  that  usually  after  the  injection  there  is  a 
marked  increase  in  the  capacity  of  the  blood  serum  to  kill  the 
typhoid  bacillus  in  vitro.  There  is  little  doubt  that  these  observa- 
tions indicate  that  the  vaccinated  person  possesses  a  degree  of 
immunity  against  the  bacillus,  and  this  conclusion  is  borne  out 
by  the  results  obtained  in  the  use  of  the  vaccine  as  a  prophy- 
lactic against  typhoid  fever.  Extensive  observations  have  been 
made  in  the  British  army  in  India,  and  in  the  South  African 
War  the  efficacy  of  the  treatment  was  put  to  test.  Though 
in  isolated  cases  not  much  difference  was  observed  among 
those  treated  as  compared  with  those  untreated,  yet  the  broad 
general  result  may  be  said  to  leave  little  doubt  that  on  the 
one  hand  protective  inoculation  diminishes  the  tendency  for  the 
individual  to  contract  typhoid  fever,  and  on  the  other,  if  the 
disease  be  contracted,  the  likelihood  of  its  having  a  fatal  result 
is  diminished.  Thus  in  India  of  4502  soldiers  inoculated,  *98 
per  cent  contracted  typhoid,  while  of  25,851  soldiers  in  the 
same  stations  who  were  not  inoculated,  2*54  per  cent  took  the 
disease.  In  Ladysmith  during  the  siege  there  were  1705 
soldiers  inoculated,  among  whom  2  per  cent  of  cases  occurred, 
and  10,529  uninoculated,  among  whom  14  per  cent  suffered 
from  typhoid.  Wright  has  collected  statistics  dealing  in  all 
with  49,600  individuals,  of  whom  8600  were  inoculated,  and 


344  TYPHOID   FEVER 

showed  a  case  incidence  of  2*25  per  cent,  with  a  case  mortality 
of  12  per  cent;  in  the  remaining  41,000  uninoculated  the  case 
incidence  was  5*75  per  cent  and  the  case  mortality  21  per  cent. 
The  best  results  seemed  to  be  obtained  when  ten  days  after  the 
first  inoculation  a  second  similar  inoculation  is  practised. 
Wright  has  found  that  in  certain  cases  immediately  after 
inoculation  there  is  a  fall  in  the  bactericidal  power  of  the  blood 
(negative  phase),  and  he  is  of  opinion  that  this  indicates  a 
temporary  increased  susceptibility  to  the  disease.  He  therefore 
recommends  that  when  possible  the  vaccination  should  be  carried 
out  some  time  previous  to  the  exposure  to  infection.  There  can 
be  very  little  doubt  that  in  this  method  an  important  prophy- 
lactic measure  has  been  discovered. 

Antityphoid  Serum.  Chantemesse  has  immunised  animals  with  dead 
cultures  of  the  typhoid  bacillus,  and  having  found  that  their  sera  had 
protective  and  curative  effects  in  other  animals,  has  used  such  sera 
in  human  cases  of  typhoid  with  apparent  good  result.  In  the  hands 
of  others,  however,  such  a  line  of  treatment  has  not  been  equally 
successful. 

Methods  of  Examination. — The  methods  of  microscopic 
examination,  and  of  isolation  of  typhoid  bacilli  from  the  spleen 
post  mortem,  have  already  been  described.  They  may  be  isolated 
from  the  Peyer's  patches,  lymphatic  glands,  etc.,  by  a  similar 
method. 

During  life,  typhoid  bacilli  may  be  obtained  in  culture  in  the 
following  ways  : — 

(a)  From  the  Spleen. — This  is  the  most  certain  method  of 
obtaining  the  typhoid  bacillus  during  the  continuance  of  a  case. 
The  skin  over  the  spleen  is  purified  and,  a  sterile  hypodermic 
syringe  being  plunged  into  the  organ,  there  is  withdrawn  from 
the  splenic  pulp  a  droplet  of  fluid,  from  which  plates  are  made. 
In  a  large  proportion  of  cases  of  typhoid  the  bacillus  may  be 
thus  obtained,  failure  only  occurring  when  the  needle  does  not 
happen  to  touch  a  bacillus.     Numerous  observations  have  shown 
that,  provided  the  needle  be  not  too  large,  the  procedure  is  quite 
safe.     Its  use,  however,  is  scarcely  called  for. 

(b)  from  the  Urine. — Typhoid  bacilli  are  present  in  the  urine 
in  at  least  twenty -five  per  cent  of  cases,  especially  late  in  the 
disease,  probably  chiefly  when  there  are  groups  in  the  kidney 
substance.     For   methods   of   examining   suspected   urine,     see 
p.  69. 

(c)  From  the  Stools. — During  the  first  ten  days  of  a  case  of 
typhoid  fever,  the  bacilli  can  be  isolated  from  the  stools  by  the 
ordinary  plate  methods — preferably  in  MacConkey's  lactose  bile 


METHODS   OF   EXAMINATION  345 

salt  neutral-red  agar,  or  in  the  medium  of  Drigalski  and  Conradi 
(v.  p.  42).  After  that  period,  though  the  continued  infectiveness 
of  the  disease  indicates  that  they  are  still  present,  their  isolation  is 
very  difficult.  We  have  seen  that  after  ulceration  is  fairly  estab- 
lished by  the  sloughing  of  the  necrosed  tissue,  the  numbers  present 
in  the  patches  are  much  diminished,  and  therefore  there  are  fewer 
cast  off  into  the  intestinal  lumen,  and  that  in  addition  there  is  a 
correspondingly  great  increase  of  the  b.  coli,  which  thus  causes 
any  typhoid  bacilli  in  a  plate  to  be  quite  outgrown.  From  the 
fact  that  the  ulcers  in  a  case  of  typhoid  may  be  very  few  in 
number,  it  is  evident  that  there  may  be  at  no  time  very  many 
typhoid  bacilli  in  the  intestine.  The  microscopic  examination 
of  the  stools  is  of  course  useless  as  a  means  of  diagnosing  the 
presence  of  the  typhoid  bacillus. 

Isolation  from  Water  Supplies. — A  great  deal  of  work  has  been  done 
on  this  subject.  It  is  evident  that  if  it  is  difficult  to  isolate  the  bacilli 
from  the  stools  it  must  a  fortiori  be  much  more  difficult  to  do  so  when 
the  latter  are  enormously  diluted  by  water.  The  b.  typhosus  has,  how- 
ever, been  isolated  from  water  during  epidemics.  This  was  done  by  Klein 
in  the  outbreaks  in  recent  years  at  Worthing  and  Kotherham.  The  b. 
coli  is,  as  might  be  expected,  the  organism  most  commonly  isolated  in 
such  circumstances.  In  the  case  of  both  bacteria,  the  whole  series  of 
culture  reactions  must  be  gone  through  before  any  particular  organism 
isolated  is  identified  as  the  one  or  the  other ;  probably  there  are  saprophytes 
existing  in  nature  which  only  differ  from  them  in  one  or  two  reactions. 
In  the  examination  of  water,  the  addition  of  '2  per  cent  carbolic  acid  to 
the  medium  inhibits  to  a  certain  extent  the  growth  of  other  bacteria, 
while  the  b.  typhosus  and  the  b.  coli  are  unaffected.  In  examining 
waters,  the  ordinary  plate  methods  are  generally  used,  but  the  Conradi- 
Drigalski  or  MacConkey  media  may  be  employed  with  advantage. 
Klein  filters  a  large  quantity  through  a  Berkefeld  filter  and,  brushing 
off  the  bacteria  retained  on  the  porcelain,  makes  cultures.  A  much 
greater  concentration  of  the  bacteria  is  thus  obtained.  From  time  to  time 
various  substances  have  been  used  with  the  object  of  inhibiting  the  growth 
of  the  b.  coli  without  interfering  with  that  of  the  b.  typhosus.  Most 
of  these  have  not  stood  the  test  of  experience.  Lately  caffeine  has  been 
used  for  this  end.  For  use  in  examining  waters  the  following  is  the 
method  employed.  To  900  c.c.  of  the  suspected  water  there  are  added 
10  grammes  nutrose  dissolved  in  80  c.c.  of  sterile  water,  and  5  grammes 
of  caffeine  dissolved  in  sterile  distilled  water  heated  to  80°  C.  and  cooled 
to  55°  C.  before  addition.  After  mixing  the  ingredients  there  is  added 
10  c.c.  of  -1  per  cent  crystal  violet.  The  flask  is  incubated  at  37°  C.  for 
12  hours  and  then  plates  of  Conradi-Drigalski  medium  are  inoculated  from 
it.  For  investigation  of  faeces,  a  medium  made  up  as  above  but  with 
ordinary  sterile  water  may  be  infected  and  a  similar  procedure  followed. 
On  the  whole  there  is  little  to  be  gained  from  this  attempt  to  isolate  the 
typhoid  bacillus  from  water  in  any  particular  case,  and  it  is  much  more 
useful  for  the  bacteriologist  to  bend  his  energies  towards  the  obtaining 
of  the  indirect  evidence  of  contamination  of  water  by  sewage,  to  the 
nature  of  which  attention  has  been  called  in  Chap.  IV. 


346  TYPHOID   FEVER 

BACTERIA  IN  DYSENTERY. 

Dysentery  has  for  long  been  recognised  as  including  a  number 
of  different  pathological  conditions,  and  within  more  recent  times 
amoebic  and  non-amoebic  forms  have  been  distinguished.  Of  the 
latter  bacteria  have  been  believed  to  be  the  causal  agents,  and  an 
organism  described  by  Shiga  in  1898  has  almost  certainly  been 
established  as  the  cause  of  a  large  proportion  of  cases.  Shiga's 
observations  were  made  in  Japan,  and  confirmatory  results  have 
been  obtained  by  Kruse  in  Germany,  by  Flexner  and  by  Strong 
and  Harvie  in  the  Philippine  Islands,  and  more  recently  by  Vedder 
and  Duval  in  the  United  States.  It  is  now  further  recognised 
that  the  epidemics  of  dysentery  which  from  time  to  time  occur 
in  lunatic  asylums  are  usually  due  to  bacilli  of  this  type,  and  in 
America  the  organism  has  been  demonstrated  in  the  summer 
diarrhoea  of  children.  The  evidence  for  the  relationship  of  the 
organism  to  the  disease  consists  chiefly  in  the  apparently  con- 
stant presence  of  the  organism  in  the  dejecta  in  this  form  of 
dysentery,  and  the  agglutination  of  the  organism  by  the  serum 
of  patients  suffering  from  the  disease,  but  confirmatory  evidence 
has  also  come  from  animal  experimentation.  From  different 
epidemics  a  great  many  different  strains  of  the  dysentery  bacillus 
have  been  obtained,  but  these  all  possess  common  characters  and 
are  undoubtedly  closely  related  to  one  another.  The  various 
strains  resolve  themselves  into  two  chief  groups,  whose  differences 
lie  in  their  behaviour  towards  certain  sugars,  in  their  capacities 
of  originating  indol  and  in  their  agglutinating  reactions.  The 
relation  of  amceba3  to  dysentery  will  be  discussed  in  the 
Appendix. 

Bacillus  Dysenterise  (Shiga). — Morphological  Cliaracters. — 
This  bacillus  morphologically  closely  resembles  the  typhoid 
bacillus,  but  is  on  the  whole  somewhat  plumper,  and  filamentous 
forms  are  comparatively  rare.  Involution  forms  sometimes  occur, 
especially  in  glucose  agar.  Most  observers  have  found  no  trace 
of  motility,  whilst  others  say  that  it  is  slightly  motile.  Vedder 
and  Duval  have,  however,  by  a  modification  of  Van  Ermengen's 
process,  demonstrated  in  the  case  of  one  strain  the  presence  of 
numerous  lateral  flagella,  which  are  of  great  fineness,  but  of 
considerable  length.  No  spore  formation  occurs ;  the  organism 
is  stained  readily  by  the  ordinary  dyes,  but  is  decolorised  by 
Gram's  method. 

Cultural  Characters.— In  these  also  considerable  resemblance 
is  presented  to  the  typhoid  bacillus.  In  gelatin  a  whitish  line  of 
growth  occurs  along  the  puncture,  but  the  superficial  film-like 


BACILLUS   DYSENTERIC  347 

growth  is  usually  absent,  or  at  least  poorly  marked.  In  plate 
cultures  also  the  superficial  growths  are  smaller  and  have  less  of 
the  film-like  character,  than  those  of  the  typhoid  organism.  On 
agar,  growth  occurs  as  a  smooth  film  with  regular  margins,  but 
after  two  or  three  days,  especially  if  the  surface  be  moist,  Vedder 
and  Duval  describe  an  outgrowth  of  lateral  offshoots  on  the 
surface  of  the  medium.  On  agar  plates  the  colonies  resemble 
those  of  the  typhoid  organism,  being  of  smaller  size  and  less 
opaque  than  those  of  the  bacillus  coli.  In  peptone  bouillon  a 
uniform  haziness  is  produced.  As  has  been  indicated,  different 
strains  of  the  bacillus  behave  differently  towards  different  sugars, 
and  the  results  of  all  observers  do  not  agree,  so  that  only  general 
statements  can  be  made.  Without  going  into  the  question  of 
the  particular  strains  to  be  placed  in  the  two  groups  we  may  say 
that,  roughly,  these  may  be  classified  into  the  Shiga-Kruse  group 
and  the  Flexner  group.  All  produce  acid  in  peptone-glucose  and 
in  taurocholate  peptone-glucose,  none  produce  change  in  lactose  or 
cane-sugar.  The  Shiga  group  do  not  produce  acid  in  maltose  or 
mannite,  while  the  Flexner  group  do,  and,  generally  speaking,  the 
former  do  not  produce  indol  while  the  latter  do.  Forms  inter- 
mediate between  the  two  groups  occur.  There  is  never  any 
evolution  of  gas  observed  in  sugar  media.  In  litmus  milk  there 
is  developed  at  first  a  slight  degree  of  acidity,  which  is  followed 
by  a  phase  of  increased  alkalinity ;  no  coagulation  of  the  milk 
ever  occurs.  On  potato  the  organism  forms  a  transparent  or 
whitish  layer,  which,  however,  in  the  course  of  a  few  days  assumes 
a  brownish-red  or  dirty  grey  colour,  with  some  discoloration  of 
the  potato  at  the  margin  of  the  growth. 

Relation  to  the  Disease. — The  organism  has  been  found  in 
large  numbers  in  the  dejecta,  especially  in  the  acute  cases,  where 
it  may  be  present  in  almost  pure  culture.  In  the  thirty-six  cases 
examined,  Shiga  obtained  it  in  thirty-four  from  the  dejecta,  and  in 
the  two  others  post  mortem  from  the  intestinal  mucous  membrane. 
The  organism  does  not  appear  to  spread  deeply  or  to  invade 
the  general  circulation.  In  the  more  chronic  cases  it  is  difficult 
to  obtain  on  account  of  the  large  number  of  the  bacillus  coli  and 
other  bacteria  present.  Vedder  and  Duval  found  agar  plates  to 
be  the  best  method  of  culture,  these  being  incubated  at  the 
blood  temperature.  They  also  found  that  if  the  colonies  which 
appeared  at  twelve  hours  were  marked  with  a  pencil,  there  was 
a  greater  probability  of  obtaining  the  bacillus  of  dysentery  from 
those  which  appeared  later,  most  of  those  appearing  early  being 
colonies  of  the  bacillus  coli.  MacConkey's  agar  medium  with 
lactose  added  may  be  used  for  isolation  from  stools.  A  little  of 


348  TYPHOID   FEVER 

the  faeces  is  rubbed  up  in  broth  and  some  of  the  mixture  stroked 
on  the  medium.  The  formation  of  acid  by  the  coli  colonies  enables 
them  to  be  excluded,  and,  therefore,  as  the  b.  dysenteric  is  not 
a  lactose  fermenter,  the  colourless  colonies  which  develop  after 
twenty-four  hours  are  picked  out  for  further  investigation. 

As  already  stated,  both  acute  and  chronic  cases  are  marked 
by  the  presence  of  this  organism.  In  the  former,  where  death 
may  occur  in  from  one  to  six  days,  the  chief  changes,  according 
to  Flexner,  are  a  marked  swelling  and  corrugation  of  the  mucous 
membrane,  with  haemorrhage  and  pseudo-membrane  at  places. 
There  is  extensive  coagulation-necrosis  with  fibrinous  exuda- 
tion and  abundance  of  polymorpho-nuclear  leucocytes,  and  the 
structure  of  the  mucous  membrane,  as  well  as  that  of  the 
muscularis  mucosae,  is  often  lost  in  the  exudation.  There  is 
also  great  thickening  of  the  sub-mucosa,  with  great  infiltration  of 
leucocytes,  these  being  chiefly  of  the  character  of  "  plasma  cells." 
In  the  more  chronic  forms  the  changes  correspond,  but  are 
more  of  a  proliferative  character.  The  mucous  membrane  is 
granular,  and  superficial  areas  are  devoid  of  epithelium,  whilst 
ulceration  and  pseudo-membrane  are  present  in  varying  degree. 
A  feature  of  bacillary  dysentery  is  the  fact  that  abscess  of  the 
liver  does  not  occur  as  a  complication. 

Agglutination. — All  the  above-mentioned  observers  agree  re- 
garding the  agglutination  of  this  bacillus  by  the  serum — that  is, 
in  the  cases  of  dysentery  from  which  the  organism  can  be  cul- 
tivated. The  reaction  may  appear  on  the  second  day,  and  is 
most  marked  after  from  six  to  seven  days  in  the  acute  cases ;  it 
is  usually  given  in  a  dilution  of  from  one  in  twenty  to  one  in 
fifty  within  an  hour,  though  sometimes  much  higher  dilutions 
give  a  positive  result.  In  the  more  chronic  cases  the  reaction 
is  less  marked,  and  here  the  sedimentation  method  is  to  be 
preferred.  It  is  difficult  to  make  any  general  statements  with 
regard  to  the  effects  of  dysenteric  sera  on  the  different  strains 
of  the  bacilli,  but  it  may  be  said  that  generally  a  serum 
agglutinates  the  strain  which  produced  it  and  the  other  strains 
of  the  same  group  in  higher  dilutions  than  it  does  the  strains  of 
the  other  group.  Many  observers  have  found  that  the  serum 
from  a  case  associated  with  strains  of  the  Shiga-Kruse  group 
have  not  agglutinated  strains  of  the  Flexner  group,  and 
corresponding  observations  have  been  made  in  cases  associated 
with  the  Flexner  group.  Often  the  sera  of  animals  immunised 
with  bacilli  have  been  used  for  such  tests,  but  apparently  great 
care  must  be  exercised  in  basing  diagnoses  on  such  observations, 
for  some  species  of  animals  when  treated  with  a  particular  strain 


BACILLUS   DYSENTERIC  349 

will  yield  a  serum  which  is  active  against  many  more  strains 
than  other  species  will  when  immunised  with  that  strain. 
Agglutination  of  the  organism  has  not  been  obtained  with  serum 
from  cases  other  than  those  of  dysentery,  nor  has  a  similar  bacillus 
been  cultivated  from  other  such  sources.  The  reaction  is  also 
absent  in  those  cases  of  dysentery  which  are  manifestly  of  amoebic 
nature. 

Pathogenic  Properties. — The  organism  is  pathogenic  in  guinea- 
pigs  and  other  laboratory  animals,  but,  in  these,  characteristic 
changes  in  the  intestine  are  often  awanting.  Shiga,  however, 
obtained  such  effects  by  introducing  the  organism  into  the 
stomach  of  young  cats  and  dogs,  and  confirmatory  results  were 
obtained  by  Flexner.  Such  attempts  have  been  specially 
successful  when  the  virulence  of  the  organism  has  been  previously 
exalted  by  intraperitoneal  passage.  In  two  cases,  apparently 
well  authenticated,  a  dysenteric  condition  has  followed  in  the 
human  subject  from  ingestion  of  pure  cultures  of  the  organism. 

It  is  probable  that  in  the  action  of  the  bacillus  a  toxin  is 
concerned.  It  has  been  found  that  the  filtrate  from  three  weeks' 
old  cultures  in  alkaline  bouillon  is  very  toxic  to  animals,  especially 
rabbits,  and  that,  however  introduced  into  the  body,  it  causes 
a  hsemorrhagic  enteritis  with  a  diphtheritic-like  exudate  on 
the  surface  of  the  mucous  membrane.  According  to  some  obser- 
vers the  toxin  is  more  readily  obtainable  from  the  Shiga-Kruse 
strains  than  from  the  Flexner  strains.  The  toxin  is  fairly  resist- 
ant to  heat,  standing  temperatures  up  to  70°  C.  without  being 
injured.  From  the  fact,  that  by  the  maceration  of  cultures 
whose  filtrates  are  relatively  non-toxic  a  stronger  poison  can  be 
obtained,  the  dysentery  toxin  has  been  thought  to  be  an 
endotoxin,  but  on  this  point  no  definite  opinion  can  be  expressed. 
Immunisation  Experiments. — Both  large  and  small  animals 
have  been  immunised  against  the  bacillus  and  also  against  its 
toxic  filtrates.  In  the  former  case  the  immunisation  has  been 
commenced  either  with  non-lethal  doses  of  living  cultures  or  with 
cultures  killed  by  heat.  The  nature  of  the  immunisation  is 
probably  complex.  When  cultures  have  been  used,  a  bactericidal 
serum,  in  which  immune  bodies  and  complements  (vide  Immunity) 
are  concerned,  is  developed.  When  the  toxin  is  used  for  immun- 
isation a  serum  protecting  against  the  toxin  is  produced.  Ac- 
cording to  some  results  animals  immunised  with  cultures  are 
immune  against  the  toxin,  and  vice  versa.  All  races  of  animal 
do  not  lend  themselves  to  immunisation.  Large  animals  (horses, 
goats)  have  been  immunised  with  the  toxin  with  a  view  of  ob- 
taining sera  for  use  in  human  dysentery,  and  in  certain  cases, 


350  TYPHOID   FEVER 

notably  in  the  work  of  Rosenthal,  a  distinct  therapeutic  effect 
has  been  produced  by  the  subcutaneous  administration  of  the 
serum,  especially  in  early  cases  of  the  disease. 

It  will  be  seen  that  the  evidence  furnished  is  practically 
conclusive  as  to  the  causal  relationship  between  this  bacillus  and 
one  form  of  dysentery,  a  form,  moreover,  which  is  both  wide- 
spread and  embraces  a  large  proportion  of  cases  of  the  disease  ; 
and  especially  of  importance  is  the  fact  that  observations  made 
independently  in  different  countries  have  yielded  practically 
identical  results  on  this  point. 

Bacillus  Dysenterise  (Ogata). — Ogata  obtained  this  bacillus  in  an 
extensive  epidemic  in  Japan  in  which  no  amoebae  were  present.  He 
found  in  sections  of  the  affected  tissues  enormous  numbers  of  small 
bacilli  of  about  the  same  thickness  as  the  tubercle  bacillus,  but  very 
much  shorter.  These  bacilli  were  sometimes  found  in  a  practically  pure 
condition.  They  were  actively  motile  and  could  be  stained  by  Gram's 
method.  He  also  obtained  pure  cultures  from  various  cases  and  tested 
their  pathogenic  effects.  They  grew  well  on  gelatin,  at  the  ordinary 
temperature  producing  liquefaction,  the  growth  somewhat  resembling 
that  of  the  cholera  spirillum.  By  injection  into  cats  and  guinea-pigs,  as 
well  as  by  feeding  them,  this  organism  was  found  to  have  distinct 
pathogenic  effects  ;  these  were  chiefly  confined  to  the  large  intestine, 
hsemorrhagic  inflammation  and  ulceration  being  produced.  It  still 
remains  to  be  determined  whether  this  organism  has  a  causal  relation- 
ship to  one  variety  of  dysentery. 

BACILLUS  ENTEIUTIDIS  SPOROGENES. 

This  organism  was  first  isolated  by  Klein  from  the  evacuations  in  an 
outbreak  of  diarrhoea  following  the  ingestion  of  milk  which  contained 
the  microbe,  and  it  was  subsequently  found  by  him  in  certain  cases  of 
infantile  diarrhea  and  of  summer  diarrhoea,  in  certain  instances  in  milk, 
and  as  a  constant  inhabitant  of  sewage  (see  Chap.  IV.).  In  films  made 
from  the  stools  in  diarrhoea  cases  where  it  is  present  it  can  be  micro- 
scopically recognised  as  a  bacillus  1'6  /*  to  4 '8  ft  in  length  and  "8  ^  in 
breadth,  staining  by  ordinary  stains  and  retaining  the  dye  in  Gram's 
method.  It  often  contains  a  spore  near  one  of  the  ends,  or  sometimes 
nearer  the  centre.  It  is  slightly  motile,  and  in  cultures  can  be  shown  to 
possess  a  small  number  of  terminal  flagella.  It  grows  well  under 
anaerobic  conditions  in  ordinary  media,  especially  on  those  containing 
reducing  agents.  On  agar  the  colonies  are  circular,  grey,  and  translucent, 
and  under  a  low  power  are  seen  to  have  a  granular  appearance.  On  this 
medium  spore  formation  does  not  occur,  but  is  easily  obtained  if  the 
organism  is  grown  on  solidified  blood  serum,  which,  further,  is  liquefied 
during  growth.  On  gelatin  plates  liquefaction  commences  after  twenty- 
four  hours  at  20°  C.  It  produces  acid  and  gas  in  bile-salt  glucose  media, 
and  in  peptone  salt  solution  containing  glucose  or  mannite.  Spore 
formation  can  be  seen  to  take  place  in  2  per  cent  dextrose  gelatin,  but 
the  degree  seems  to  be  in  inverse  ratio  to  the  amount  of  gas  formation. 
Very  typical  is  the  growth  on  milk,  and  it  is  by  this  medium  that 
isolation  can  be  best  effected.  A  small  quantity  of  the  material 


SUMMER   DIARRHCEA  351 

suspected  to  contain  the  bacillus  is  placed  in  15  to  20  c.c.  of  sterile 
milk,  which  is  then  heated  for  ten  minutes  at  80°  C.  to  destroy  all 
vegetative  bacteria  ;  the  tube  is  cooled,  placed  under  anaerobic  con- 
ditions, and  incubated  at  37°  C.  for  from  twenty-four  to  thirty-six  hours. 
If  the  bacillus  be  present  there  is  abundant  gas  formation,  and  almost 
complete  separation  of  the  curd  from  the  whey  takes  place.  The  former 
adheres  to  the  sides  of  the  tube  in  shreds,  and  large  masses  gather  with 
the  cream  on  the  top  of  the  fluid,  all  being  torn  by  the  gas  evolved. 
The  whey  is  only  slightly  turbid  and  contains  numerous  bacilli.  The 
growth  has  an  odour  of  butyric  acid.  If  a  small  quantity  (say  1  c.c.)  of 
the  whey  be  injected  into  a  guinea-pig,  the  animal  becomes  ill  in  a  few 
hours  and  dies  in  twenty-four  hours.  At  the  point  of  inoculation  the 
skin  and  subcutaneous  tissues,  and  sometimes  even  the  subjacent  muscles, 
are  green  and  gangrenous  and  evil-smelling,  there  is  considerable  oedema, 
and  there  may  also  be  gas  formation.  The  exudation  is  crowded  with 
bacilli,  which,  however,  are  not  generally  distributed  in  any  numbers 
throughout  the  body.  These  pathogenic  properties  of  the  bacillus 
enteritidis  sporogenes  are  important  in  its  recognition,  for  its  culture 
reactions  taken  alone  are  very  similar  to  those  of  the  bacillus  butyricus 
of  Botkin. 

SUMMEK    DlARRH(EA. 

As  lias  been  already  stated,  both  the  bacillus  of  dysentery,  b.  coli 
and  the  b.  enteritidis  sporogenes  have  been  found  associated  with 
epidemics  of  this  disease.  This  indicates  that  the  condition  may 
be  originated  by  a  variety  of  organisms,  and  it  is  further  probable 
that  the  clinical  features  in  different  epidemics  vary.  The 
multiple  origin  of  the  disease  has  been  illustrated  by  the  work 
of  Morgan,  who,  in  a  careful  investigation  of  the  disease  in 
Britain,  has  been  unable  to  find  evidence  of  the  dysentery 
bacillus  being  present.  He  has,  however,  very  constantly  found 
in  the  stools  and  intestine  a  bacillus  ("  Morgan's  No.  1  bacillus  ") 
which  is  a  motile  Gram-negative  organism  producing  acid  and 
slight  gas  formation  in  glucose,  Isevulose,  and  galactose,  and  no 
change  in  mannite,  dulcite,  maltose,  dextrin,  cane  sugar,  lactose, 
inulin,  amygdalin,  salicin,  arabinose,  rafrinose,  sorbite,  or  ery- 
thrite ;  it  further  causes  indol  formation,  and  in  litmus  milk 
slowly  originates  an  alkaline  reaction.  It  produces  diarrhoea  and 
death  in  young  rabbits,  rats,  and  monkeys  when  these  animals 
are  fed  on  cultures.  It  is  thus  possible  that  in  this  bacillus 
we  have  still  another  cause  of  the  disease. 


CHAPTEE 

DIPHTHERIA. 

THERE  is  no  better  example  of  the  valuable  contributions  of 
bacteriology  to  scientific  medicine  than  that  afforded  in  the  case 
of  diphtheria.  Not  only  has  research  supplied,  as  in  the  case  of 
tubercle,  a  means  of  distinguishing  true  diphtheria  from  condi- 
tions which  resemble  it,  but  the  study  of  the  toxins  of  the 
bacillus  has  explained  the  manner  by  which  the  pathological 
changes  and  characteristic  symptoms  of  the  disease  are  brought 
about,  and  has  led  to  the  discovery  of  the  most  efficient  means 
of  treatment,  namely,  the  anti- diphtheritic  serum. 

Historical. — The  first  account  of  the  bacillus  now  known  to  be  the 
cause  of  diphtheria  was  given  by  Klebs  in  1883,  who  described  its 
characters  in  the  false  membrane,  but  made  no  cultivations.  It  was 
first  cultivated  by  Loffler  from  a  number  of  cases  of  diphtheria,  his 
observations  being  published  in  1884,  and  to  him  we  owe  the  first 
account  of  its  characters  in  cultures  and  of  some  of  its  pathogenic  effects 
on  animals.  The  organism  is  for  these  reasons  known  as  the  Klebs- 
Loffler  bacillus,  or  simply  as  Lbffler's  bacillus.  By  experimental  in- 
oculation with  the  cultures  obtained,  Loffler  was  able  to  produce  false 
membrane  on  damaged  mucous  surfaces,  but  he  hesitated  to  conclude 
definitely  that  this  organism  was  the  cause  of  the  disease,  for  he  did 
not  find  it  in  all  the  cases  of  diphtheria  examined,  he  was  not  able  to 
produce  paralytic  phenomena  in  animals  by  its  injection,  and,  further, 
he  obtained  the  same  organism  from  the  throat  of  a  healthy  child.  This 
organism  became  the  subject  of  much  inquiry,  but  its  relationship  to 
the  disease  may  be  said  to  have  been  definitely  established  by  the 
brilliant  researches  of  Roux  and  Yersin  which  showed  that  the  most 
important  features  of  the  disease  could  be  produced  by  means  of  the 
separated  toxins  of  the  organism.  Their  experiments  were  published  in 
1888-90.  Further  light  has  been  thrown  on  the  subject  by  the  work  of 
Sidney  Martin,  who  has  found  that  there  can  be  separated  from  the 
organs  in  cases  of  diphtheria  substances  which'  act  as  nerve  poisons,  and 
also  produce  other  phenomena  met  with  in  diphtheria. 

General  Facts. — Without  giving  a  description  of  the  patho- 
logical changes  in  diphtheria,  it  will  be  well  to  mention  the  out- 

352 


BACILLUS   DIPHTHERIA  353 

standing  features  which  ought  to  be  considered  in  connection 
with  its  bacteriology.  In  addition  to  the  formation  of  false 
membrane,  which  may  prove  fatal  by  mechanical  effects,  the 
chief  clinical  phenomena  are  the  symptoms  of  general  poisoning, 
great  muscular  weakness,  tendency  to  syncope,  and  albuminuria ; 
also  the  striking  paralyses  which  occur  later  in  the  disease, 
and  which  may  affect  the  muscles  of  the  pharynx,  larynx,  and 
eye,  or  less  frequently  the  lower  limbs  (being  sometimes  of 
paraplegic  type),  all  these  being  grouped  together  under  the 
term  "  post-diphtheritic  paralyses'."  It  may  be  stated  here  that  all 
these  conditions  have  been  experimentally  reproduced  by  the 
action  of  the  bacillus  of  diphtheria,  or  by  its  toxins.  Other 
bacteria  are,  however,  concerned  in  producing  various  secondary 
inflammatory  complications  in  the  region  of  the  throat,  such  as 
ulceration,  gangrenous  change,  and  suppuration,  which  may  be 
accompanied  by  symptoms  of  general  septic  poisoning.  The  detec- 
tion of  the  bacillus  of  Loffler  in  the  false  membrane  or  secretions 
of  the  mouth  is  to  be  regarded  as  supplying  the  only  certain 
means  of  diagnosis  of  diphtheria. 

Bacillus  Diphtherise. — Microscopical  Characters. — If  a  film 
preparation  be  made  from  a  piece  of  diphtheria  membrane  (in 
the  manner  described  below)  and  stained  with  methylene-blue, 
the  bacilli  are  found  to  have  the  following  characters.  They  are 
slender  rods,  straight  or  slightly  curved,  and  usually  about  3  p. 
in  length,  their  thickness  being  a  little  greater  than  that  of 
the  tubercle  bacillus.  The  size,  however,  varies  somewhat  in 
different  cases,  and  for  this  reason  varieties  have  been  distin- 
guished as  small  and  large,  and  even  of  intermediate  size.  It  is 
sufficient  to  mention  here  that  in  some  cases  most  are  about  3  //. 
in  length,  whilst  in  others  they  may  measure  fully  5  /x.  Corre- 
sponding differences  in  size  are  found  in  cultures.  They  stain 
deeply  with  the  blue,  sometimes  being  uniformly  coloured,  but 
often  showing,  in  their  substance,  little  granules  more  darkly 
stained,  so  that  a  dotted  or  beaded  appearance  is  presented. 
Sometimes  the  ends  are  swollen  and  more  darkly  stained  than 
the  rest;  often,  however,  they  are  rather  tapered  off  (Fig.  116). 
In  some  cases  the  terminal  swelling  is  very  marked,  so  as  to 
amount  to  clubbing,  and  with  some  specimens  of  methylene-blue 
these  swellings  and  granules  stain  of  a  violet  tint.  Distinct 
clubbing,  however,  is  less  frequent  than  in  cultures.  There  is  a 
want  of  uniformity  in  the  appearance  of  the  bacilli  when  com- 
pared side  by  side.  They  usually  lie  irregularly  scattered  or  in 
clusters,  the  individual  bacilli  being  disposed  in  all  directions. 
Some  may  be  contained  within  leucocytes.  They  do  not  form 
23 


354  DIPHTHERIA 

chains,  but  occasionally  forms  longer  than  those  mentioned  may 
be  found,  and  these  specially  occur  in  the  spaces  between'  the 
fibrin  as  seen  in  sections. 

Distribution  of  the  Bacillus. — The  diphtheria  bacillus  may 
be  found  in  the  membrane  wherever  it  is  formed,  and  may  also 
occur  in  the  secretions  of  the  pharynx  and  larynx  in  the  disease. 


v  / 

/ 

**  , 

9    - 

*  'V-" 

FIG.  116. — Film  preparation  from  diphtheria  membrane  ;  showing  numerous 
diphtheria  bacilli.      One  or  two  degenerated  forms  are  seen  near  the 
centre  of  the  field.     (Cultures  made  from  the  same  piece  of  membrane 
showed  the  organism  to  be  present  in  practically  pure  condition.) 
Stained  with  methylene-blue.      x  1000. 

It  may  be  mentioned  that  distinctions  formerly  drawn  between 
true  diphtheria  and  non-diphtheritic  conditions  from  the  appear- 
ance and  site  of  the  membrane,  have  no  scientific  value,  the  only 
true  criterion  being  the  presence  of  the  diphtheria  bacillus.  The 
occurrence  of  a  membranous  formation  produced  by  streptococci 
has  already  been  mentioned  (p.  184). 

In  diphtheria  the  membrane  has  a  somewhat  different 
structure  according  as  it  is  formed  on  the  surface  covered  with 
stratified  squamous  epithelium  as  in  the  pharynx,  or  on  a  surface 


DISTRIBUTION   OF   THE   BACILLUS 


355 


covered  by  ciliated  epithelium  as  in  the  trachea.  In  the  former 
situation  necrosis  of  the  epithelium  occurs  either  uniformly  or  in 
patches,  and  along  with  this  there  is  marked  inflammatory 
reaction  in  the  connective  tissue  beneath,  attended  by  abundant 
fibrinous  exudation.  The  necrosed  epithelium  becomes  raised 
up  by  the  fibrin,  and  its  interstices  are  also  filled  by  it.  The 


FIG.  117. — Section  through  a  diphtheritic  membrane  in  trachea,  showing  diph- 
theria bacilli  (stained  darkly)  in  clumps,  and  also  scattered  amongst  the 
librin.  Some  streptococci  are  also  shown,  towards  the  surface  on  the 
left  side.  . 

Stained  by  Gram's  method  and  Bismarck-brown,      x  1 000. 

fibrinous  exudation  also  occurs  around  the  vessels  in  the  tissue 
beneath,  and  in  this  way  the  membrane  is  firmly  adherent.  In 
the  trachea,  on  the  other  hand,  the  epithelial  cells  rapidly 
become  shed,  and  the  membrane  is  found  to  consist  almost 
exclusively  of  fibrin  with  leucocytes,  the  former  arranged  in  a 
reticulated  or  somewhat  laminated  manner,  and  varying  in 
density  in  different  parts.  The  membrane  lies  upon  the  base- 
ment membrane,  and  is  less  firmly  adherent  than  in  the  case  of 
the  pharynx 


356  DIPHTHERIA 

The  position  of  the  diphtheria  bacilli  varies  somewhat  in 
different  cases,  but  they  are  most  frequently  found  lying  in  oval 
or  irregular  clumps  in  the  spaces  between  the  fibrin,  towards  the 
superficial,  that  is,  usually,  the  oldest  part  of  the  false  membrane 
(Fig.  117).  There  they  may  be  in  a  practically  pure  condition, 
though  streptococci  and  occasionally  some  other  organisms  may 
be  present  along  with  them.  They  may  occur  also  deeper,  but 
are  rarely  found  in  the  fibrin  around  the  blood  vessels.  On  the 
surface  of  the  membrane  they  may  be  also  seen  lying  in  large 
numbers,  but  are  there  accompanied  by  numerous  other 
organisms.  Occasionally  a  few  bacilli  have  been  detected  in  the 
lymphatic  glands.  As  Loffler  first  described,  they  may  be 
found  after  death  in  pneumonic  patches  in  the  lung,  these 
being  due  to  a  secondary  extension  by  the  air  passages.  They 
have  also  been  occasionally  found  in  the  spleen,  liver,  and 
other  organs  after  death.  This  occurrence  is  probably  to 
be  explained  by  an  entrance  into  the  blood  stream  shortly 
before  death,  similar  to  what  occurs  in  the  case  of  other 
organisms,  e.g.  the  bacillus  coli  communis.  The  diphtheria 
bacillus  may  also  infect  other  mucous  membranes.  It  is  found 
in  true  diphtheria  of  the  conjunctiva,  and  may  also  occur 
in  similar  affections  of  the  vulva  and  vagina :  some  of  these 
cases  have  been  treated  successfully  with  diphtheria  antitoxin. 
The  pseudo-diphtheria  bacillus,  however,  may  also  occur  in  these 
situations. 

Association  with  other  Organisms. — The  diphtheria  organism  is 
sometimes  present  alone  in  the  membrane,  but  more  frequently 
is  associated  with  some  of  the  pyogenic  organisms,  the  strepto- 
coccus pyogenes  being  the  commonest.  The  staphylococci,  and 
occasionally  the  pneumococcus  or  the  bacillus  coli,  may  be 
present  in  some  cases.  Streptococci  are  often  found  lying  side 
by  side  with  the  diphtheria  bacilli  in  the  membrane,  and  also 
penetrating  more  deeply  into  the  tissues.  In  some  cases  of 
tracheal  diphtheria,  we  have  found  streptococci  alone  at  a  lower 
level  in  the  trachea  than  the  diphtheria  bacilli,  where  the 
membrane  was  thinner  and  softer,  the  appearance  in  these  cases 
being  as  if  the  streptococci  acted  as  exciters  of  inflammation  and 
prepared  the  way  for  the  bacilli.  It  is  still  a  matter  of  dispute 
as  to  whether  the  association  of  the  diphtheria  bacillus  with  the 
pyogenic  organisms  is  a  favourable  sign  or  the  contrary,  though 
on  experimental  grounds  the  latter  is  the  more  probable.  We 
know,  however,  that  some  of  the  complications  of  diphtheria 
may  be  due  to  the  action  of  pyogenic  organisms.  The  extensive 
swelling  of  the  tissues  of  the  neck,  sometimes  attended  by 


CULTIVATION   OF   THE   BACILLUS 


357 


FIG.  118.  —  Cultures  of  the 
diphtheria  bacillus  on  an 
agar  plate  ;  twenty  -  six 
hours'  growth. 

(a)  Two  successive  strokes ;  (b) 
isolated  colonies  from  the  same 
plate. 


suppuration  in  the  glands,  and  also  various  hsemorrhagic  con- 
ditions, have  been  found  to  be  as- 
sociated with  their  presence  ;  in  fact, 
in  some  cases  the  diphtheritic  lesion 
enables  them  to  get  a  foothold  in 
the  tissues,  where  they  exert  their 
usual  action  and  may  lead  to  exten- 
sive suppurative  change,  to  septic 
poisoning  or  to  septicaemia.  In  cases 
where  a  gangrenous  process  is  super- 
added,  a  great  variety  of  organisms 
may  be  present,  some  of  them  being 
anaerobic.  Against  such  complica- 
tions produced  by  other  organisms 
anti-diphtheritic  serum  produce  no 
favourable  effect. 

Cultivation.  —  The  diphtheria 
bacillus  grows  best  in  cultures  at  the 
temperature  of  the  body;  growth 
still  takes  place  at  22°  C.,  but  ceases 
about  20°  C.  The  best  media  are  the 
following  :  Lomer's  original  medium 

(p.  40),  solidified  blood   serum,  alkaline  blood  serum  (Lorrain 

Smith),  blood  agar,  and 
the  ordinary  agar  media. 
If  inoculations  be  made 
on  the  surface  of  blood 
serum  with  a  piece  of 
*•§*  diphtheria  membranes, 
^^iT"3^^  '  Vi  c°l°nies  °f  tne  bacillus 

*V^  JS^    may    appear    in    twelve 

.    hours  and  are  well  formed 

«^.  ^v     ^     <  \    within  twenty-four  hours 

^&£P*»J$P  often    before    any   other 

^J^§AJMr*  growths  are  visible.     The 

colonies  are  small  circular 
discs  of  opaque  whitish 
colour,  their  centre  being 
thicker  and  of  darker 

Fie,  119.-Diphtheria  bacilli  from  a  twenty-    «^  appearance  when 
four  hours' culture  on  agar.  Vlewed     %     transmitted 

Stained  with  methylene-blue.      x  1000.        light  than  the  periphery. 

On  the  second  or  third 
day  they  may  reach  3  mm.  in  size,  but  when  numerous  they 


358 


DIPHTHERIA 


remain  smaller.      On  the  agar  media  the  colonies  have  much 
__  the  same  appearance  (Fig. 

'  -AL*  11 7)  but  grow  less  quickly, 

^\!r\f  an(i  sometimes  they  may 

be  comparatively  minute, 
£.  V       so  as  rather  to  resemble 
those  of  the  streptococcus 
pyogenes.     In  stroke  cul- 
tures the  growth  forms  a 
continuous   layer   of    the 
same  dull  whitish  colour, 
the  margins  of  which  often 
show  single  colonies  partly 
or   completely   separated. 
On   gelatin  at    22°   C.  a 
puncture  culture  shows  a 
line    of    dots   along    the 
Fig.   120. — Diphtheria  bacilli  of  larger,  size   needle  track,  whilst  at  the 
than  in  previous  figure,  showing  also  ir-    surface  a  small  disc  forms, 
regular  staining  of  protoplasm.     From  a    rather 


thicker 


the 


three  days'  agar  culture.  "  tmuKer      in 

Stained  with  weak  carbol-fuchsin.     x  1000.    middle.      In  none  of  the 

media  does  any  liquefac- 
tion occur.  In  bouillon  the  organism  produces  a  turbidity 
which  soon  settles  to  the 
bottom  and  forms  a  pow- 
dery layer  on  the  wall  of 
the  vessel.  By  starting 
the  growth  on  the  surface 
and  keeping  the  flasks  at 
rest  a  distinct  scum  forms, 
and  this  is  especially  suit- 
able for  the  development 
of  toxin.  Ordinary  bouillon 
becomes  acid  during  the 
first  two  or  three  days, 
and  several  days  later 
again  acquires  an  alkaline 
reaction.  If,  however,  the 
bouillon  is  glucose -free 
(p.  75)  the  acid  reaction  Fig- 121.— Involution  forms  of  the  diphtheria 

does  not  occur.  5acil,lus  ;  5°m  an  a^ar  culture  of  seven 

T    ,1  ,.    ,,     .       .,,.          days  growth. 

In  these  media  the  bacilli  stained  with  carbol-thionin-blue.      x  1000. 
show  the  same  characters 
as  in  the  membrane,  but  the  irregularity  in  staining  is  more  marked 


POWERS   OF   RESISTANCE   OF   BACILLUS      359 

(Figs.  119,  120).  They  are  at  first  fairly  uniform  in  size  and 
shape,  but  later  involution  forms  are  present.  Many  are  swollen 
at  their  ends  into  club-shaped  masses  which  stain  deeply,  and  the 
protoplasm  becomes  broken  up  into  globules  with  unstained 
parts  between  (Fig.  121).  Some  become  thicker  throughout, 
and  segmented  so  as  to  appear  like  large  cocci,  and  others  show 
globules  at  their  ends,  the  rest  of  the  rod  appearing  as  a  faintly- 
stained  line.  Occasionally  branched  forms  are  met  with.  The 
bacilli  are  non-motile,  and  do  not  form  spores. 

Staining. — They  take  up  the  basic  aniline  dyes,  e.g.  methy- 
lene-blue  in  watery  solution,  with  great  readiness,  and  stain 
deeply,  the  granules  often  giving  the  metachromatic  reaction  as 
described.  They  also  retain  the  colour  in  Gram's  method, 
though  they  are  more  easily  decolorised  than  the  pyogenic  cocci. 
By  Neisser's  stain  (p.  108)  the  granules  are  stained  almost  black, 
the  rest  of  the  bacillary  substance  yellowish  brown. 

Powers  of  Resistance,  etc. — In  cultures  the  bacilli  possess 
long  duration  of  life  ;  at  the  room  temperature  they  may  survive 
for  two  months  or  longer.  In  the  moist  condition,  whether  in 
cultures  or  in  membrane,  they  have  a  low  power  of  resistance, 
being  killed  at  60°  C.  in  a  fewr  minutes.  On  the  other  hand,  in 
the  dry  condition  they  have  great  powers  of  endurance.  In 
membrane  which  is  perfectly  dry,  for  example,  they  can  resist  a 
temperature  of  98°  C.  for  an  hour.  Dried  diphtheria  membrane, 
kept  in  the  absence  of  light  and  at  the  room  temperature,  has 
been  proved  to  contain  diphtheria  bacilli  still  living  and  virulent 
at  the  end  of  several  months.  The  presence  of  light,  moisture, 
or  a  higher  temperature,  causes  them  to  die  out  more  rapidly. 
Corresponding  results  have  been  obtained  with  bacilli  obtained 
from  cultures  and  kept  on  dried  threads.  These  facts,  especially 
with  regard  to  drying,  are  of  great  importance,  as  they  show  that 
the  contagium  of  diphtheria  may  be  preserved  for  a  long  time 
in  the  dried  membrane. 

Effects  of  Inoculation. — In  considering  the  effects  produced 
in  animals  by  experimental  inoculations  of  pure  cultures,  we 
have  to  keep  in  view  the  local  changes  which  occur  in  diphtheria, 
and  also  the  symptoms  of  general  poisoning. 

As  Loffler  stated  in  his  original  paper  inoculation  of  the 
healthy  mucous  membranes  of  various  animals  with  pure  cultures 
causes  no  lesion,  but  the  formation  of  false  membrane  may 
result  when  the  surface  is  injured  by  scarification  or  otherwise. 
A  similar  result  may  be  obtained  when  the  trachea  is  inoculated 
after  tracheotomy  has  been  performed.  In  this  case  the 
surrounding  tissues  may  become  the  seat  of  a  blood-stained 


360  DIPHTHEEIA 

oedema,  and  the  lymphatic  glands  become  enlarged,  the  general 
picture  resembling  pretty  closely  that  of  laryngeal  diphtheria. 
The  membrane  produced  by  such  experiments  is  usually  less 
firm  than  in  human  diphtheria,  and  the  bacilli  in  the  membrane 
are  less  numerous.  Rabbits  inoculated  after  tracheotomy  often 
die,  and  Roux  and  Yersin  were  the  first  to  observe  that  in  some 
cases  paralysis  may  appear  before  death. 

Subcutaneous  injection  in  guinea-pigs,  of  diphtheria  bacilli  in 
a  suitable  dose,  produces  death  within  thirty-six  hours.  At  the 
site  of  inoculation  there  is  usually  a  small  patch  of  greyish 
membrane,  whilst  in  the  tissues  around  there  is  extensive 
inflammatory  oedema,  often  associated  with  haemorrhages,  and 
there  is  also  some  swelling  of  the  corresponding  lymphatic  glands. 
The  internal  organs  show  general  congestion,  the  suprarenal 
capsules  being  especially  reddened  and  often  showing  haemorrhage, 
The  renal  epithelium  may  show  cloudy  swelling,  and  there  is 
often  effusion  into  the  pleural  cavities.  After  injection  the 
bacilli  increase  in  number  for  a  few  hours,  but  multiplication 
soon  ceases,  and  at  the  time  of  death  they  may  be  less  numerous 
than  when  injected.  The  bacilli  remain  practically  local, 
cultures  made  from  the  blood  and  internal  organs  giving  usually 
negative  results,  though  sometimes  a  few  colonies  may  be 
obtained.  If  a  non-fatal  dose  of  a  culture  be  injected,  a  local 
necrosis  of  the  skin  and  subcutaneous  tissue  may  follow  at  the 
site  of  inoculation. 

In  rabbits,  after  subcutaneous  inoculation,  results  of  the  same 
nature  follow,  but  these  animals  are  less  susceptible  than  guinea- 
pigs,  and  the  dose  requires  to  be  proportionately  larger.  Roux 
and  Yersin  found  that  after  intravenous  injection  the  bacilli 
rapidly  disappeared  from  the  blood,  and  when  1  c.c.  of  a  broth 
culture  had  been  injected  no  trace  of  the  organisms  could  be 
detected  by  culture  after  twenty-four  hours :  nevertheless  the 
animals  died  with  symptoms  of  general  toxaemia,  nephritis  also 
being  often  present  (cf.  Cholera,  p.  408).  The  dog  and  sheep 
are  also  susceptible  to  inoculation  with  virulent  bacilli,  but  the 
mouse  and  rat  enjoy  a  high  degree  of  immunity. 

Klein  found  that  cats  also  were  susceptible  to  inoculation.  The 
animals  usually  die  after  a  few  days,  and  post  mortem  there  is  well-marked 
nephritis.  He  also  found  that  after  subcutaneous  injection  in  cows,  a 
vesicular  eruption  appeared  on  the  teats  of  the  udder,  tbe  fluid  in  which 
contained  diphtheria  bacilli.  At  the  time  of  death  the  diphtheria  bacilli 
were  still  alive  and  virulent  at  the  site  of  injection.  The  most  striking 
result  of  these  experiments  is  that  the  diphtheria  bacilli  passed  into  the 
circulation  and  were  present  in  the  eruption  on  the  udder.  He  considers 
that  this  may  throw  light  on  certain  epidemics  of  diphtheria  in  which 


THE   TOXINS   OF   DIPHTHERIA  361 

the  contagion  was  apparently  carried  by  the  milk.  Other  observers 
have,  however,  failed  to  obtain  similar  results.  Dean  and  Todd,  in 
investigating  an  outbreak  of  diphtheria  traceable  to  milk  supplied, 
found  a  vesicular  eruption  on  the  teats  of  the  udder  in  which  diphtheria 
bacilli  were  present.  They,  however,  came  to  the  conclusion  that  these 
bacilli  were  not  the  cause  of  the  eruption,  but  were  the  result  of  a 
secondary  contamination,  probably  from  the  saliva  of  the  milkers.  The 
occurrence  of  a  true  infection  with  the  diphtheria  bacillus  in  the  horse 
has  been  described  by  Cobbett. 

The  Toxins  of  Diphtheria. — As  in  the  above  experiments 
the  symptoms  of  poisoning  and  ultimately  a  fatal  result  occur 
when  the  bacilli  are  diminishing  in  number,  or  even  after  they 
have  practically  disappeared,  Roux  and  Yersiri  inferred  that  the 
chief  effects  were  produced  by  toxins,  and  this  supposition  they 
proved  to  be  correct.  They  showed  that  broth  cultures  of  three 
or  four  weeks'  growth  freed  from  bacilli  by  filtration  were  highly 
toxic.  The  filtrate  when  injected  into  guinea-pigs  and  other 
animals  produces  practically  the  same  effects  as  the  living  bacilli ; 
locally  there  is  fibrinous  exudation  but  a  considerable  amount 
of  inflammatory  oedema,  and,  if  the  animal  survive  long  enough, 
necrosis  in  varying  degree  of  the  superficial  tissues  may  follow. 
The  toxicity  may  be  so  great  that  *01  c.c.  or  even  less  may  be 
fatal  to  a  guinea-pig  in  twenty-four  hours. 

After  injection  either  of  the  toxin  or  of  the  living  bacilli, 
when  the  animals  survive  long  enough,  paralytic  phenomena 
may  occur.  The  hind  limbs  are  usually  affected  first,  the 
paralysis  afterwards  extending  to  other  parts,  though  sometimes 
the  fore-limbs  and  neck  first  show  the  condition.  Sometimes 
symptoms  of  paralysis  do  not  appear  till  two  or  three  weeks 
after  inoculation.  After  paralysis  has  appeared,  a  fatal  result 
usually  follows  in  the  smaller  animals,  but  in  dogs  recovery  may 
take  place.  There  is  evidence  that  these  paralytic  phenomena 
are  produced  by  toxone  (p.  171),  as  they  may  occur  when  there 
is  injected  along  with  the  toxin  sufficient  antitoxin  to  neutralise 
the  more  rapidly  acting  toxin  proper.  This  toxone  is  supposed 
by  Ehrlich  to  have  a  different  toxic  action,  i.e.  a  different 
toxophorous  group,  from  that  of  the  toxin,  and  to  have  a  weaker 
affinity  for  antitoxin  ;  much  of  it  may  thus  be  left  unneutralised. 
It  is  to  be  noted  in  this  connection  that  paralytic  symptoms  are 
of  not  uncommon  occurrence  in  the  human  subject  after  treatment 
with  antitoxin,  the  explanation  of  which  occurrence  is  probably 
the  same  as  that  just  given.  One  point  of  much  interest  is  the 
high  degree  of  resistance  to  the  toxin  possessed  by  mice  and 
rats.  Roux  and  Yersin,  for  example,  found  that  2  c.c.  of  toxin, 
which  was  sufficient  to  kill  a  rabbit  in  sixty  hours,  had  no  effect 


362  DIPHTHERIA 

on  a  mouse,  whilst  of  this  toxin  even  ^  c.c.  produced  extensive 
necrosis  of  the  skin  of  the  guinea-pig. 

Preparation  of  the  Toxin. — The  obtaining  of  a  very  active 
toxin  in  large  quantities  is  an  essential  in  the  preparation  of  anti- 
diphtheritic  serum.  Certain  conditions  favour  the  development 
of  a  high  degree  of  toxicity,  viz.,  a  free  supply  of  oxygen,  the 
presence  of  a  large  proportion  of  peptone  or  albumin  in  the 
medium,  and  the  absence  of  substances  which  produce  an  acid 
reaction.  In  the  earlier  work  a  current  of  sterile  air  was  made 
to  pass  over  the  surface  of  the  medium,  as  it  was  found  that  by 
this  means  the  period  of  acid  reaction  was  shortened  and  the 
toxin  formation  favoured.  This  expedient  is  now  considered 
unnecessary  if  an  alkaline  medium  free  from  glucose  is  used,  as 
in  this  no  acid  reaction  is  developed  :  it  is  then  sufficient  to 
grow  the  cultures  in  shallow  flasks.  The  absence  of  glucose — an 
all-important  point — may  be  attained  by  the  method  described 
above  (p.  75),  or  by  using  for  the  preparation  of  the  meat  extract 
flesh  which  is  just  commencing  to  putrefy  (Spronck).  L.  Martin 
uses  a  medium  composed  of  equal  parts  of  freshly  prepared 
peptone  (by  digesting  pigs'  stomachs  with  HC1  at  35°  C.),  and 
glucose-free  veal  bouillon.  By  this  medium  he  has  obtained  a 
toxin  of  which  y^  c.c.  is  the  fatal  dose  to  a  guinea-pig  of  500 
grms.  He  finds  that  glucose,  glycerin,  saccharose,  and  galactose 
lead  to  the  production  of  an  acid  reaction,  whilst  glycogen  does 
not.  The  latter  fact  explains  how  some  observers  have  found 
that  bouillon  prepared  from  quite  fresh  flesh  is  suitable  for  toxin 
formation.  There  is  in  all  cases  a  period  at  which  the  toxicity 
reaches  a  maximum,  usually  in  2-3  weeks,  but  earlier  if  the  toxin 
is  rapidly  formed  ;  later  the  toxicity  diminishes.  Martin  found 
that  in  his  medium  the  maximum  was  reached  on  the  8th-10th 
day.  It  may  be  added  that  the  power  of  toxin  formation 
varies  much  in  different  races  of  the  diphtheria  bacillus,  and 
that  many  may  require  to  be  tested  ere  one  suitable  is  obtained. 

Properties  and  Nature  of  the  Toxin. — The  toxic  substance  in 
filtered  cultures  is  a  relatively  unstable  body.  When  kept  in 
sealed  tubes  in  the  absence  of  light,  it  may  preserve  its  powers 
little  altered  for  several  months,  but.  on  the  other  hand,  it 
gradually  loses  them  when  exposed  to  the  action  of  light  and 
air.  As  will  be  shown  later  (p.  472),  the  toxin  probably  does  not 
become  destroyed,  but  its  toxophorous  group  suffers  a  sort  of 
deterioration  so  that  a  toxoid  is  formed  which  has  still  the 
power  of  combining  with  antitoxins.  Heating  at  58°  C.  for 
two  hours  destroys  the  toxic  properties  in  great  part,  but  not 
altogether.  When,  however,  the  toxin  is  evaporated  to  dryness,  it 


NATURE   OF   THE   TOXIN  363 

has  much  greater  resistance  to  heat.  One  striking  fact,  discovered 
by  Roux  and  Yersin,  is  that  after  an  organic  acid,  such  as  tartaric 
acid,  is  added  to  the  toxin  the  toxic  property  disappears,  but  that 
it  can  be  in  great  part  restored  by  again  making  the  fluid  alkaline. 

Guinochet  showed  that  toxin  was  formed  by  the  bacilli  when 
grown  in  urine  with  no  proteid  bodies  present.  After  growth 
had  taken  place  he  could  not  detect  proteid  bodies  in  the  fluid, 
but  on  account  of  the  very  minute  amount  of  toxin  present, 
their  absence  could  not  be  excluded.  Uschinsky  also  found  that 
toxic  bodies  were  produced  by  diphtheria  bacilli  when  grown  in 
a  proteid-free  medium.1  It  follows  from  this  that  if  the  toxin  is 
a  proteid,  it  may  be  formed  by  synthesis  within  the  bodies  of  the 
bacilli.  Brieger  and  Boer  have  separated  from  diphtheria  cultures 
a  toxic  body  which  gives  no  proteid  reaction  (vide  p.  166). 

Toxic  bodies  have  also  been  obtained  from  the  tissues  of  those 
who  have  died  from  diphtheria.  Roux  and  Yersin,  by  using 
a  filtered  watery  extract  from  the  spleen  from  very  virulent  cases 
of  diphtheria,  produced  in  animals  death  after  wasting  and 
paralysis,  and  also  obtained  similar  results  by  employing  the 
urine.  The  subject  of  toxic  bodies  in  the  tissues  has,  however, 
been  specially  worked  out  by  Sidney  Martin.  He  has  separated 
from  the  tissues,  and  especially  from  the  spleen,  of  patients  who 
have  died  from  diphtheria,  by  precipitation  with  alcohol,  chemical 
substances  of  two  kinds,  namely,  albumoses  (proto-  and  deutero-, 
but  especially  the  latter),  and  an  organic  acid.  The  albumoses 
when  injected  into  rabbits,  especially  in  repeated  doses,  produce 
fever,  diarrhoea,  paresis,  and  loss  of  weight,  with  ultimately  a 
fatal  result.  As  in  the  experiments  with  the  toxin  from  cultures, 
the  posterior  limbs  are  first  affected  ;  afterwards  the  respiratory 
muscles,  and  finally  the  heart,  are  implicated.  He  further  found 
that  this  paresis  is  due  to  well-marked  changes  in  the  nerves. 
The  medullary  sheaths  first  become  affected,  breaking  up  into 
globules;  ultimately  the  axis  cylinders  are  involved,  and  may 
break  across,  so  that  degeneration  occurs  in  the  peripheral 
portion  of  the  nerve  fibres.  Such  changes  occur  irregularly  in 
patches,  both  sensory  and  motor  fibres  being  affected.  Fatty 
change  takes  place  in  the  associated  muscle  fibres.  There  may 
also  be  a  similar  condition  in  the  cardiac  muscle.  The  organic 
acid  has  a  similar  but  weaker  action.  Substances  obtained  from 
diphtheria  membrane  have  an  action  like  that  of  the  bodies 

1  Uschinsky 's  medium  has  the  following  composition  :  water,  1000  parts  ; 
glycerin,    30-40  ;  sodium    chloride,  5-7  ;  calcium    chloride,  "1  ;  magnesium 
sulphate,    '2- '4  ;    di-potassium  phosphate,  '2- '25  ;  ammonium  lactate,  6-7;- 
sodium  asparaginate,  3-4. 


364  DIPHTHERIA 

obtained  from  the  spleen,  but  in  higher  degree.  Martin  con- 
siders that  this  is  due  to  the  presence  in  the  membrane  of  an 
enzyme  which  has  a  proteolytic  action  within  the  body,  resulting 
in  the  formation  of  poisonous  albumoses.  According  to  this 
view  the  actually  toxic  bodies  are  not  the  direct  product  of  the 
bacillus,  but  are  formed  by  the  enzyme  which  is  produced  by  it 
locally  in  the  membrane.  Cartwright  Wood  has  also  found  that 
when  diphtheria  cultures  in  an  albumin-containing  medium  are 
filtered  germ-free  and  exposed  to  65°  C.  for  an  hour  (the  supposed 
ferments  being  thus  destroyed),  there  still  remain  albumoses 
which  produce  febrile  reaction  and  are  active  in  developing 
immunity.  In  the  present  state  of  knowledge  we  are  not  in  a 
position  to  give  an  interpretation  of  such  experiments,  and  we 
cannot  even  say  whether  the  proteids  obtained  by  precipitation 
from  cultures  and  from  the  tissues  are  in  themselves  toxic,  or 
whether  the  true  toxic  bodies  are  carried  down  along  with 
them. 

Immunity. — This  is  described  in  the  general  chapter  on 
Immunity.  It  is  sufficient  to  state  here  that  a  high  degree  of 
immunity,  against  both  the  bacilli  and  their  toxins,  can  be 
produced  in  various  animals  by  gradually  increasing  doses  either 
of  the  bacilli  or  of  their  filtered  toxins  (vide  Chap.  XIX.). 

Variations  in  the  Virulence  of  the  Diphtheria  Bacillus. — In 
cultures  on  serum  the  diphtheria  bacilli  retain  their  virulence 
fairly  well,  but  they  lose  it  much  more  quickly  .on  less  suitable 
media,  such  as  glycerin  agar.  Roux  and  Yersin  found  that, 
when  the  bacilli  were  grown  at  an  abnormally  high  temperature, 
namely,  39*5°  C.,  and  in  a  current  of  air,  the  virulence  diminished 
so  much  that  they  became  practically  innocuous.  When  the 
virulence  was  much  diminished,  these  observers  found  that  it 
could  be  restored  if  the  bacilli  were  inoculated  into  animals  along 
with  streptococci,  inoculation  of  the  bacilli  alone  not  being 
successful  for  this  purpose.  If,  however,  the  virulence  had  fallen 
very  low,  even  the  presence  of  the  streptococci  was  insufficient 
to  restore  it.  As  a  rule,  the  cultures  most  virulent  to  guinea- 
pigs  are  obtained  from  the  gravest  cases  of  diphtheria,  though  to 
this  rule  there  are  frequent  exceptions.  ,  Perhaps  the  majority  of 
observers  have  found  that  the  bacilli  of  the  larger  form  are 
usually  more  virulent  than  those  of  the  shorter  form ;  but  this  is 
not  invariably  the  case,  as  sometimes  short  forms  are  obtained 
which  possess  an  extremely  virulent  character.  It  has  been 
abundantly  established  that  after  the  cure  of  the  disease,  the 
bacilli  may  persist  in  the  mouth  for  weeks,  though  they  often 
quickly  disappear.  Roux  and  Yersin  found,  by  making  cultures 


BACILLI  ALLIED   TO   DIPHTHERIA   BACILLUS    365 

at  various  stages  after  the  termination  of  the  disease,  that  these 
bacilli  in  the  mouth  gradually  become  attenuated. 

L.  Martin,  moreover,  has  shown  that  some  races  of  diphtheria 
bacillus  are  so  attenuated  that  1  c.c.  of  a  24  hours'  growth 
in  bouillon  does  not  cause  death  in  a  guinea-pig,  yet  their  true 
nature  is  shown  not  only  by  their  microscopical  characters,  etc., 
but  also  by  the  fact  that  on  more  prolonged  growth  they  form 
small  quantities  of  toxin,  which  is  neutralised  by  diphtheria 
antitoxin.  The  persistence  of  these  non-virulent  bacilli  in  the 
throats  of  those  who  have  suffered  from  the  disease,  and  their 
occasional  presence  in  quite  healthy  individuals,  may  manifestly 
be  of  importance  in  relation  to  the  continuance  of  the  infection 
and  the  reappearance  of  epidemics  of  the  disease. 

Bacilli  allied  to  the  Diphtheria  Bacillus. 

Bacteriological  examinations  carried  on  within  recent  times 
have  shown  that  the  diphtheria  bacillus  is  merely  a  member  of  a 
group  of  organisms  with  closely  allied  characters  which  are  of 
common  occurrence  and  have  a  wide  distribution.  The  terms 
"  pseudo-diphtheria  bacilli "  and  "  diphtheroid  bacilli "  have  been 
applied  in  a  loose  way  to  organisms  which  resemble  the  diphtheria 
bacillus  microscopically,  especially  as  regards  the  beaded  ap- 
pearance. Such  bacilli  have  been  obtained  from  the  mouth, 
nose,  skin,  genital  organs,  and  even  from  the  blood  in  certain 
diseases.  They  are  to  be  met  with  sometimes  in  conditions  of 
health,  and  they  have  been  obtained  from  many  diverse  morbid 
conditions — from  skin  diseases,  from  coryza,  from  leprosy,  and 
even  from  general  paralysis  of  the  insane.  As  has  been  found 
with  other  groups  the  differentiation  is  a  matter  of  considerable 
difficulty.  Some  are  practically  identical  with  the  diphtheria 
bacillus  both  morphologically  and  culturally,  and  a  few  even  give 
the  characteristic  reaction  with  Neisser's  stain ;  others  again 
differ  in  essential  particulars.  The  fermentative  action  on 
sugars  *  has  also  been  called  into  requisition  as  a  means  of  distin- 
guishing them,  but  the  results  obtained  cannot  be  said  to  be  of  a 
definite  character,  and  further  work  is  necessary.  It  may  be 
stated,  however,  that  most  observers  have  found  the  diphtheria 
bacillus  of  all  the  members  of  the  group  to  be  the  most  active 
acid -producer,  though  here  the  difference  seems  to  be  one  of 
degree  rather  than  of  kind.  The  absence  of  the  power  of 
fermenting  certain  sugars,  notably  glucose,  may,  however,  be 
accepted  in  any  particular  case  as  sufficient  to  exclude  the 
1  Vide  a  paper  by  Graham- Smith,  Journal  of  Hygiene,  vi.  286. 


366  DIPHTHERIA 

organism  from  being  the  diphtheria  bacillus.  From  these  facts, 
and  from  what  has  been  stated  with  regard  to  attenuated 
diphtheria  bacilli,  it  will  be  seen  that  an  absolute  decision  as  to 
the  nature  of  a  suspected  organism  may  in  some  cases  be  a 
practical  impossibility.  It  may  be  that  some  of  the  "  diphtheroid  " 
organisms  cultivated  have  really  been  non-virulent  diphtheria 
bacilli.  The  bearing  of  this  on  the  practical  means  of  diagnosis 
will  be  discussed  below. 

The  term  "  pseudo-diphtheria  bacillus  "  is  often  restricted  by 
present  writers  to  an  organism  frequently  met  with  in  the  throat. 
This  organism,  which  is  also  known  as  Hofmann's  bacillus,  merits 
a  separate  description. 

Hofmann's  Bacillus  —  Pseudo  -  Diphtheria  Bacillus. — This 
organism,  described  by  Hofmann  in  1888,  is  probably  the 

same  as  one  observed  by 
Loffler  in  the  previous 
year,  and  regarded  by  him 
as  being  a  distinct  species 
from  the  diphtheria  bacil- 
lus. The  organism  is  a 
shorter  bacillus  than  the 
diphtheria  bacillus,  with 
usually  a  single  unstained 
septum  running  across  it, 
though  sometimes  there 
may  be  more  than  one 
(Fig.  122).  The  typical 
beaded  appearance  is  rarely 
seen,  and  the  reaction  with 
Neisser's  stain  is  not  given. 
FIG.  122.— Pseudo-diphtheria  bacillus  It  grows  readily  on  the 
(Hofmann's).  Young  agar  culture.  same  media  as  the  diph- 

Stained  with  thionin-blue.      x  1000.  theria    bacillus,     but     the 

colonies    are   whiter    and 

more  opaque.  It  does  not  form  acid  from  glucose  or  other 
sugars,  and  is  non-pathogenic  to  the  guinea-pig.  Involution 
forms  may  sometimes  be  produced  by  it.  It  is  usually  a  com- 
paratively easy  matter  to  distinguish,  this  organism  from  the 
diphtheria  bacillus. 

Hofmann's  bacillus  is  of  comparatively  common  occurrence  in 
the  throat  in  normal  as  well  as  diseased  conditions,  including 
diphtheria  ;  it  seems  to  be  specially  frequent  in  poorly  nourished 
children  of  the  lower  classes.  Cobbett  found  it  157  times  in  an 
examination  of  692  persons  examined,  of  whom  650  were  not 


SUMMAKY  367 

suffering  from  diphtheria.  Boycott's  statistics  show  that  the 
time  of  its  maximum  seasonal  prevalence  precedes  that  of  the 
diphtheria  bacillus.  To  what  extent,  if  any,  it  is  responsible  for 
pathological  changes  in  the  throat,  must  be  considered  a  question 
which  is  not  yet  settled.  Hewlett  and  Knight  have  found 
evidence  that  a  true  diphtheria  bacillus  may  assume  the  characters 
of  Hofmann's  bacillus,  but  attempts  to  effect  the  transformation 
have  met  with  negative  results  in  the  hands  of  other  observers. 
The  general  opinion  is  that  the  two  organisms  are  distinct  species 
which  are  comparatively  easily  distinguished  characters. 

Xerosis  Bacillus. — This  term  has  been  given  to  an  organism  first 
observed  by  Kuschbert  and  Neisser  in  xerosis  of  the  conjunctiva,  and 
which  has  been  since  found  in  many  other  affections  of  the  conjunctiva 
and  also  in  normal  conditions.  Morphologically  it  is  practically  similar 
to  the  diphtheria  bacillus,  and  even  in  cultures  presents  very  minor 
differences.  It  is,  however,  non- virulent  to  animals,  and  does  not  pro- 
duce an  acid  reaction  in  sugar-containing  bouillon,  or  does  so  to  only 
a  slight  extent ;  in  this  way  it  can  be  distinguished  from  the  diphtheria 
bacillus.  Its  morphological  characters  are  shown  in  Fig.  123. 

Action  of  the  Diphtheria  Bacillus — Summary. — From  a 
study  of  the  morbid  changes  in  diphtheria  and  of  the  results 
produced  experimentally 

by   the    bacillus   and    its  -    *jf 

toxins,  the  following  sum-  ffi^' 

mary  may  be  given  of  its 
action  in  the  body.  Locally,  v**1  it 

the  bacillus  produces   in-  ^\ 

flammatory    change    with 
fibrinous    exudation,    but  j>  *~ 

at  the  same  time  cellular  *  >*    <fr 

necrosis    is   also   an   out-          'j» 
standing  feature.     Though      ^  4, 
false  membranes  have  not        ijfj 
been     produced     by     the 
toxins,   a   necrotic    action 
may  result  when  these  are  -£*£- 

injected      subcutaneously.       Fm>  123._Xerosis  bacillus  from  a  young 
The  toxins  also  act  upon  agar  culture,      x  1000. 

the     blood  -  vessels,     and 

hence  oedema  and  tendency  to  haemorrhage  are  produced ; 
this  action  on  the  vessels  is  also  exemplified  by  the  general 
congestion  of  organs.  The  hyaline  change  in  the  walls  of 
arterioles  and  capillaries  so  often  met  with  in  diphtheria  is 
another  example  of  the  action  of  the  toxin.  The  toxins  have 


368  DIPHTHERIA 

also  a  pernicious  action  on  highly-developed  cells  and  on  nerve 
fibres.  Thus  in  the  kidney,  cloudy  swelling  occurs,  which  may 
be  followed  by  actual  necrosis  of  the  secreting  cells,  and  along 
with  these  changes  albuminuria  is  present.  The  action  is  also 
well  seen  in  the  case  of  the  muscle  fibres  of  the  heart,  which 
may  undergo  a  sort  of  hyaline  change,  followed  by  granular  dis- 
integration or  by  an  actual  fatty  degeneration.  These  changes 
are  of  great  importance  in  relation  to  heart  failure  in  the  disease. 
Changes  of  a  somewhat  similar  nature  have  been  recently 
observed  in  the  nerve  cells  of  the  central  nervous  system,  those 
lying  near  the  capillaries,  it  is  said,  being  affected  first.  There 
is  also  the  striking  change  in  the  peripheral  nerves,  which  is 
shown  first  by  the  disintegration  of  the  medullary  sheaths  as 
already  described.  It  is,  however,  still  a  matter  of  dispute  to 
what  extent  these  nerve  lesions  are  of  primary  nature  or 
secondary  to  changes  in  the  nerve  cells. 

Methods  of  Diagnosis. — The  bacteriological  diagnosis  of 
diphtheria  depends  on  the  discovery  of  the  bacillus.  As  the 
bacillus  occurs  in  largest  numbers  in  the  membrane,  a  portion  of 
this  should  be  obtained  whenever  it  is  possible,  and  transferred 
to  a  sterile  test-tube.  (The  tube  can  be  readily  sterilised  by 
boiling  some  water  in  it.).  If,  however,  membrane  cannot  be 
obtained,  a  scraping  of  the  surface  with  a  platinum  loop  may  be 
sufficient.  Where  the  membrane  is  confined  to  the  trachea  the 
bacilli  are  often  present  in  the  secretions  of  the  pharynx,  and 
may  be  obtained  from  that  situation  by  swabbing  it  with  cotton- 
wool (non-antiseptic),  the  swab  being  put  into  a  sterile  tube  or 
bottle  for  transport.  A  convenient  method  is  to  twist  a  piece  of 
cotton-wool  round  the  roughened  end  of  a  piece  of  very  stout 
iron  wire,  six  inches  long,  and  pass  the  other  end  of  the  latter 
through  a  cotton  plug  inserted  in  the  mouth  of  a  test-tube 
(compare  Fig.  48,  the  wire  taking  the  place  of  the  pipette),  and 
sterilise.  In  use  the  wire  and  plug  are  extracted  in  one  piece, 
and  after  swabbing  are  replaced  in  the  tube  for  transit.  A 
scraping  may  be  made  off  the  swab  for  microscopic  examination, 
and  the  swab  may  be  smeared  over  the  surface  of  a  serum  tube 
to  obtain  a  culture.  This  method  of  taking  and  treating  swabs 
is  that  usually  employed  in  routine  public  health  work.  The 
results  obtained  ordinarily  suffice  for  the  diagnosis  of  cases 
suspected  to  be  diphtheritic  in  nature. 

The  means  for  identifying  the  bacillus  are  (a)  By  micro- 
scopical examination. — For  microscopical  examination  it  is 
sufficient  to  tease  out  a  piece  of  the  membrane  with  forceps  and 
rub  it  on  a  cover-glass ;  if  it  be  somewhat  dry  a  small  drop  of 


METHODS   OF   DIAGNOSIS  369 

normal  saline  should  be  added.  The  films  are  then  dried  in  the 
usual  way  and  stained  with  any  ordinary  basic  stain,  though 
methylene-blue  is  on  the  whole  to  be  preferred,  used  either  as 
a  saturated  watery  solution  or  in  the  form  of  Loffler's  solution. 
After  staining  for  two  or  three  minutes  the  films  are  washed  in 
water,  dried,  and  mounted.  As  a  rule  no  decolorising  is 
necessary,  as  the  blue  does  not  overstain.  Neisser's  stain  (p.  108) 
may  also  be  used  with  advantage.  Any  secretion  from  the 
pharynx  or  other  part  is  to  be  treated  in  the  same  way.  The 
value  of  microscopical  examination  alone  depends  much  upon 
the  experience  of  the  observer.  In  some  cases  the  bacilli  are 
present  in  characteristic  form  in  such  numbers  as  to  leave  no 
doubt  in  the  matter.  In  other  cases  a  few  only  may  be  found, 
mixed  with  large  quantities  of  other  organisms,  and  sometimes 
their  characters  are  not  sufficiently  distinct  to  render  a  definite 
opinion  possible.  We  have  frequently  obtained  the  bacillus  by 
means  of  cultures,  when  the  result  of  microscopical  examination 
of  the  same  piece  of  membrane  was  non-conclusive.  As  already 
said,  however,  microscopical  examination  alone  is  more  reliable 
after  the  observer  has  had  experience  in  examining  cases  of 
diphtheria  and  making  cultures  from  them. 

(6)  By  making  Cultures. — For  this  purpose  a  piece  of  the 
membrane  should  be  separated  by  forceps  from  the  pharynx  or 
other  part  when  that  is  possible.  It  should  be  then  washed 
well  in  a  tube  containing  sterile  water,  most  of  the  surface  im- 
purities being  removed  in  this  way.  A  fragment  is  then  fixed  in 
a  platinum  loop  by  means  of  sterile  forceps,  and  a  series  of 
stroke  cultures  are  made  on  the  surface  of  any  of  the  media 
mentioned  (p.  357),  the  same  portion  of  the  membrane  being 
always  brought  into  contact  with  the  surface.  The  tubes  are 
then  placed  in  the  incubator  at  37°  C.,  and,  in  the  case 
of  the  serum  media  and  blood-agar,  the  circular  colonies 
of  the  diphtheria  bacillus  are  well  formed  within  twenty-four 
hours.  A  small  portion  of  a  colony  is  then  removed  by  means 
of  a  platinum  needle,  stained,  and  examined  in  the  usual  way, 
Neisser's  stain  being  also  applied.  When  the  material  has  been 
taken  from  the  throat,  an  organism  with  all  the  morphological 
and  cultural  characters  of  the  diphtheria  bacillus  may  for  all 
practical  purposes  be  accepted  as  the  diphtheria  bacillus. 

In  cases  where  a  suspicion  arises  that  the  organism  found 
is  a  pseudo-diphtheria  bacillus,  bouillon  containing  a  trace 
of  glucose  should  be  inoculated  and  incubated  at  37°  C. 
The  reaction  should  be  tested  after  one  and  after  two  days' 
growth.  If  it  remains  alkaline,  the  diphtheria  bacillus  may  be 
24 


370  DIPHTHEKIA 

excluded.  If  an  acid  reaction  results,  then  all  the  microscopical 
and  cultural  characters  must  be  carefully  observed,  and  the 
virulence  of  the  bacillus  may  be  ascertained  by  inoculating  a 
guinea-pig,  say  with  1  c.c.  of  a  broth  culture  of  two  days'  growth. 
(See  also  pp.  359,  365.)  A  fatal  result  with  characteristic 
appearances  may  be  taken  as  positive  evidence,  but  if  the  animal 
survive  there  is  still  theoretically  the  possibility  that  the 
organism  is  an  attenuated  diphtheria  bacillus  (p.  364), 


CHAPTER  XVI. 

TETANUS.1 

SYNONYMS. LOCKJAW.       GERMAN,    WUNDSTAKRKKAMPF. 

FRENCH,    TETANOS. 

Introductory. — Tetanus  is  a  disease  which  in  natural  conditions 
affects  chiefly  man  and  the  horse.  Clinically  it  is  characterised 
by  the  gradual  onset  of  general  stiffness  and  spasms  of  the  volun- 
tary muscles,  commencing  in  those  of  the  jaw  and  the  back  of  the 
neck,  and  extending  to  all  the  muscles  of  the  body.  These  spasms 
are  of  a  tonic  nature,  and,  as  the  disease  advances,  succeed  each 
other  with  only  a  slight  intermission  of  time.  There  are  often, 
towards  the  end  of  a  case,  fever  and  rise  of  respiration  and  pulse 
rate.  The  disease  is  usually  associated  with  a  wound  received  from 
four  to  fourteen  days  previously,  and  which  has  been  defiled  by 
earth  or  dung.  Such  a  wound  may  be  very  small.  The  disease 
is,  in  the  majority  of  cases,  fatal.  Post  mortem  there  is  little  to 
be  observed  on  naked -eye  examination.  The  most  marked 
feature  is  the  occurrence  of  patches  of  congestion  in  the  spinal 
cord,  and  especially  the  medulla. 

Historical.— The  general  association  of  the  development  of  tetanus  with 
the  presence  of  wounds,  though  these  might  be  very  small,  suggested  that 
some  infection  took  place  through  the  latter,  but  for  long  nothing  was 
known  as  to  the  nature  of  this  infection.  Carle  and  Rattone  in  1884 
announced  that  they  had  produced  the  disease  in  a  number  of  animals  by 
inoculation  with  material  from  a  wound  in  tetanus.  They  thus  demon- 
strated the  transmissibility  of  the  disease.  Nocolaier  (1885)  infected  mice 
and  rabbits  with  garden  earth,  and  found  that  many  of  them  developed 
tetanus.  Suppuration  occurred  in  the  neighbourhood  of  the  point  of 

1  This  disease  is  not  to  be  confused  with  the  "  tetany  "  of  infants,  which  in 
its  essential  pathology  probably  differs  from  tetanus  (vide  Frankl-Hochwart, 
"Die  Tetanic  der  Erwachsenen, "  Vienna,  1907).  This  remark  of  course  does 
not  exclude  the  possibility  of  the  occurrence  of  true  tetanus  in  very  young 
subjects. 

371 


372  TETANUS 

inoculation,  and  in  this  pus,  besides  other  organisms,  there  was  always 
present,  when  tetanus  had  occurred,  a  bacillus  having  certain  constant 
microscopic  characters.  Inoculation  of  fresh  animals  with  such  pus 
reproduced  the  disease.  Nicolaier's  attempts  at  its  isolation  by  the 
ordinary  gelatin  plate-culture  method  were,  however,  unsuccessful.  He 
succeeded  in  getting  it  to  grow  in  liquid  blood  serum,  but  always  in 
mixture  with  other  organisms.  Infection  of  animals  with  such  a  culture 
produced  the  disease.  These  results  were  confirmed  by  Rosenbach,  who, 
though  failing  to  obtain  a  pure  culture,  cultivated  the  other  organisms 
present,  and  inoculated  them,  but  with  negative  results.  He  further 
pointed  out,  as  characteristic  of  the  bacillus,  its  development  of  terminal 
spores.  In  1889,  Kitasato  succeeded  in  isolating  from  the  local  suppura- 
tion of  mice  inoculated  from  a  human  case,  several  bacilli,  only  one  of 
which,  when  injected  in  pure  culture  into  animals,  caused  the  disease, 
and  which  was  now  named  the  b.  tetani.  This  organism  is  the  same  as 
that  observed  by  Nicolaier  and  Rosenbach.  Kitasato  found  that  the 
cause  of  earlier  culture  failures  was  the  fact  that  it  could  only  grow  in  the 
absence  of  oxygen.  The  pathology  of  the  disease  was  further  elucidated 
by  Faber,  who,  having  isolated  bacterium-free  poisons  from  cultures, 
reproduced  the  symptoms  of  the  disease. 

Bacillus  Tetani. — Tf  in  a  case  of  tetanus  naturally  arising  in 
man,  there  be  a  definite  wound  with  pus  formation  or  necrotic 
change,  the  bacillus  tetani  may  be  recognised  in  film  preparations 
from  the  pus,  if  the  characteristic  spore  formation  has  occurred 
(Fig.  124).  If,  however,  the  tetanus  bacilli  have  not  formed 
spores,  they  appear  as  somewhat  slender  rods,  without  present- 
ing any  characteristic  features.  There  is  usually  present  in  such 
pus  a  great  variety  of  other  organisms — cocci  and  bacilli.  The 
characters  of  the  bacillus  are,  therefore,  bes£  studied  in  cultures. 
It  is  then  seen  to  be  a  slender  organism,  usually  about  4  p  to  5  /A 
in  length  and  *4  /x,  in  thickness,  with  somewhat  rounded  ends. 
Besides  occurring  as  short  rods  it  also  develops  filamentous 
forms,  the  latter  being  more  common  in  fluid  media.  It  stains 
readily  by  any  of  the  usual  stains  and  also  by  Gram's  method. 
A  feature  in  it  is  the  uniformity  with  which  the  protoplasm  stains. 
It  is  very  slightly  motile,  and  its  motility  can  be  best  studied  in 
an  anaerobic  hanging- drop  preparation  (p.  64).  When  stained 
by  the  special  methods  already  described,  it  is  found  to  possess 
numerous  delicate  flagella  attached  both  at  the  sides  and  at  the 
ends  (Fig.  125).  These  flagella,  though  they  maybe  of  consider- 
able length,  are  usually  curled  up  close  to  the  body  of  the  bacillus. 
The  formation  of  flagella  can  be  best  studied  in  preparations 
made  from  surface  anaerobic  culture's  (p.  62).  As  is  the  case 
with  many  other  anaerobic  flagellated  bacteria  the  flagella,  on 
becoming  detached,  often  become  massed  together  in  the  form 
of  spirals  of  striking  appearance  (Fig.  126).  At  incubation 
temperature  b.  tetani  readily  forms  spores,  and  then  presents  a 


BACILLUS   TETANI 


373 


very  characteristic  appearance.  The  spores  are  round,  and  in 
diameter  may  be  three  or  four  times  the  thickness  of  the  bacilli. 
They  are  developed  at  one  end  of  a  bacillus,  which  thus  assumes 
what  is  usually  described  as  the  drumstick  form  (Figs.  124,  127). 
In  a  specimen  stained  with  a  watery  solution  of  gentian-violet  or 
methylene-blue,  the  spores  are  uncoloured  except  at  the  periphery, 


FIG.  124. — Film  preparation  of  discharge  from  wound  in  a  case  of  tetanus, 
showing  several  tetanus  bacilli  of  "drumstick"  form.  (The  thicker 
bacillus  with  oval  and  not  quite  terminal  spore,  in  the  upper  part  of  the  field 
towards  the  right  side,  is  not  a  tetanus  bacillus  but  a  putrefactive 
anaerobe  which  was  obtained  in  pure  culture  from  the  wound.) 
Stained  with  gentian -violet.  x  1000. 

so  that  the  appearance  of  a  small  ring  is  produced  ;  if  a  powerful 
stain  such  as  carbol-fuchsin  be  applied  for  some  time,  the  spores 
become  deeply  coloured  like  the  bacilli.  Further,  especially 
if  the  preparation  be  heated,  many  spores  may  become  free  from 
the  bacilli  in  which  they  were  formed. 

Isolation. — The  isolation  of  the  tetanus  bacillus  is  somewhat 
difficult.  By  inoculation  experiments  in  animals,  its  natural 
habitat  has  been  proved  to  be  garden  soil,  and  especially  the 


374 


TETANUS 


contents  of  dung -heaps,  where  it  probably  leads  a  saprophytic 
existence,  though  its  function  as  a  saprophyte  is  unknown. 
From  such  sources  and  from  the  pus  of  wounds  in  tetanus, 
occurring  naturally  or  experimentally  produced,  it  has  been 
isolated  by  means  of  the  methods  appropriate  for  anaerobic 
bacteria.  The  best  methods  for  dealing  with  such  pus  are  as 
follows  : — 

(1)  The  principle  is  to  take  advantage  of  the  resistance  of 


FIG.  125. — Tetanus  bacilli,  showing  flagella. 
Stained  by  Rd.  Muir's  method.      x  1000. 

the  spores  of  the  bacillus  to  heat.  A  sloped  tube  of  inspissated 
serum  or  a  deep  tube  of  glucose  agar  is  inoculated  with  the 
pus  and  incubated  at  37°  C.  for  forty-eight  hours,  at  the  end  of 
which  time  numerous  spore-bearing  bacilli  can  often  be  observed 
microscopically.  The  culture  is  then  kept  at  80°  C.  for  from 
three-quarters  to  one  hour,  with  the  view  of  killing  all  organisms 
except  those  which  have  spored.  A  loopful  is  then  added  to 
glucose  gelatin,  and  roll-tube  cultures  are  made  in  the  usual 
way  and  kept  in  an  atmosphere  of  hydrogen  at  22°  C. ;  after 
five  days  the  plates  are  ready  for  examination.  Kitasato  com- 


ISOLATION   OF   THE   BACILLUS 


375 


pares  the  colonies  in  gelatin  plates  to  those  of  the  b.  subtilis. 

They  consist  of  a  thick 

centre  with  shoots  radi-  H^^w 

ating  out  on   all   sides. 

They  liquefy  the  gelatin 

more  slowly  than  the  b. 

subtilis.      This   method 

of  isolation  is  not  always 

successful,  partly  because 

along  with  the  tetanus 

bacilli,      both      in      its 

natural  habitats  outside 

the  body  and  in  the  pus 

of  wounds,  other  spore- 
forming  obligatory  and 

facultative  anaerobes  oc- 

cur,  which   grow  faster 

than  thetetanus  bacillus,        ^  126._Spiral  composed  of  numerous 

and  thus  overgrow  it.  twisted  flagella  of  the  tetanus  bacillus. 

(2)    If    in    any    dis-        Stained  by  Rd.  Muir's  method.      x  1000. 

charge  the  spore-bearing 

tetanus   bacilli    be    seen   on    microscopic   examination,   then   a 

method  of  isolation 
based  on  the  same  prin- 
ciple as  the  last  may 
be  adopted.  Inocula- 
tions with  the  suspected 
material  are  made  in 
half  a  dozen  deep  tubes 
of  glucose  agar,  previ- 
ously melted  and  kept  at 
a  temperature  of  100e 
C.  After  inoculation 
they  are  again  placed  in 
boiling  water  and  kept 
for  varying  times,  say 
for  half  a  minute,  for 
one,  three,  four,  five, 

FIG.  127. — Tetanus  bacilli ;  some  of  which  and  six  minutes  respect- 
possess  spores.  From  a  culture  in  glucose  ive}v  They  are  then 
agar,  incubated  for  three  days  at  37°  C.  •>  •>  •  -,  -, 

Stained  with  carbol-fuchsin.      x  1000.  Ponged    in    cold    water 

till  cool,  and  thereafter 

placed   in  the  incubator  at    37°  C.,    in  the  hope  that   in   one 

or  other  of  the  tubes  all  the  organisms  present  will  have  been 


376 


TETANUS 


killed,  except   the   tetanus  spores  which   can  develop  in   pure 
culture. 

(3)    Some   method  of  anaerobically  making  plates,  such  as 
that  of  Bulloch,  may  be  employed.     The  isolation  of  the  tetanus 
bacillus  is  in  many  cases  a  difficult  matter, 
and  various  expedients  require  to  be  tried. 

Characters  of  Cultures. — Pure  cultures 
having  been  obtained,  sub-cultures  can  be 
made  in  deep  upright  glucose  gelatin  or 
agar  tubes.  On  glucose  gelatin  in  such  a 
tube  there  commences,  an  inch  or  so  below 
the  surface,  a  growth  consisting  of  fine 
straight  threads,  rather  longer  in  the  lower 
than  in  the  upper  parts  of  the  tube,  radiating 
out  from  the  needle  track  (Fig.  128).  Slow 
liquefaction  of  the  gelatin  takes  place,  with 
slight  gas  formation.  In  agar  the  growth  is 
somewhat  similar,  consisting  of  small  nodules 
along  the  needle  track,  with  irregular  short 
offshoots  passing  out  into  the  medium  (Fig. 
131,  A).  There  is  slight  formation  of  gas, 
but,  of  course,  no  liquefaction.  Growth  also 
occurs  in  blood  serum  and  also  in  glucose 
bouillon  under  anaerobic  conditions.  The 
latter  is  the  medium  usually  employed  for 
obtaining  the  soluble  products  of  the  or- 
ganism. There  is  in  it  at  first  a  slight 
turbidity,  and  later  a  thin  layer  of  a 
powdery  deposit  on  the  walls  of  the  vessel. 
FIG.  128,-Stab  cul-  A?1  ^  (niltures  give  out  a  peculiar  burnt 
ture  of  the  tetanus  odour  of  rather  unpleasant  character, 
bacillus  in  glucose  Conditions  of  Growth,  etc. — The  b. 
gelatin,  showing  tetani  grows  best  at  37°  C.  The  minimum 
the  lateral  shoots  g^^h  temperature  is  about  14°  C.,  and 
(Kitasato).  .Natural  Y  -,  rtno  ^  ,,  ,  ,  ,  , 

gize  below  22    C.  growth  takes  place  very  slowly. 

Growth  takes  place  only  in  the  absence  of 
oxygen,  the  organism  being  a  strict  anaerobe.  Sporulation  may 
commence  at  the  end  of  twenty-four  hours  in  cultures  grown 
at  37°  C., — much  later  at  lower  temperatures.  Like  other 
spores,  those  of  tetanus  are  extremely  resistant.  They  can 
usually  withstand  boiling  for  five  minutes,  and  can  be  kept  in 
a  dry  condition  for  many  months  without  being  killed  or  losing 
their  virulence.  They  have  also  high  powers  of  resistance  to 
antiseptics. 


PATHOGENIC   EFFECTS  377 

Pathogenic  Effects. — The  proof  that  the  b.  tetani  is  the  cause 
of  tetanus  is  complete.  It  can  be  isolated  in  pure  culture,  and 
when  re-injected  in  pure  culture  it  reproduces  the  disease.  It 
may  be  impossible  to  isolate  it  from  some  cases  of  the  disease, 
but  the  cause  of  this  very  probably  is  the  small  numbers  in 
which  it  sometimes  occurs. 

(a)  The  Disease  as  arising  Naturally. — The  disease  occurs 
naturally,  chiefly  in  horses  and  in  man.  Other  animals  may, 
however,  be  affected.  There  is  usually  some  wound,  often  of  a 
ragged  character,  which  has  either  been  made  by  an  object 
soiled  with  earth  or  dung,  or  which  has  become  contaminated 
with  these  substances.  There  is  often  purulent  or  foetid  dis- 
charge, though  this  may  be  absent.  Microscopic  examination 
of  sections  may  show  at  the  edges  of  the  wound  necrosed  tissue 
in  which  the  tetanus  bacilli  may  -be  very  numerous.  If  a 
scraping  from  the  wound  be  examined  microscopically,  bacilli 
resembling  the  tetanus  bacillus  may  be  recognised.  If  these 
have  spored,  there  can  be  practically  no  doubt  as  to  their 
identity,  as  the  drumstick  appearance  which  the  terminal  spore 
gives  to  the  bacillus  is  not  common  among  other  bacilli.  Care 
must  be  taken,  however,  to  distinguish  it  from  other  thicker 
bacilli  with  oval  spores  placed  at  a  short  distance  from  their  ex- 
tremities, such  forms  being  common  in  earth,  etc.,  and  also  met 
with  in  contaminated  wounds  (Fig.  124).  It  is  important  to  note 
that  the  wound  through  which  infection  has  taken  place  may  be 
very  small,  in  fact,  may  consist  of  a  mere  abrasion.  In  some 
cases,  especially  in  the  tropics,  it  may  be  merely  the  bite  of  an 
insect.  The  absence  of  a  definite  channel  of  infection  has  given 
.  rise  to  the  term  "  idiopathic "  tetanus.  There  is,  however, 
practically  no  doubt  that  all  such  cases  are  true  cases  of  tetanus, 
and  that  in  all  of  them  the  cause  is  the  b.  tetani.  The  latter 
has  also  been  found  in  the  bronchial  mucous  membrane  in  some 
cases  of  the  so-called  rheumatic  tetanus,  the  cause  of  which  is 
usually  said  to  be  cold. 

The  pathological  changes  found  post  mortem  are  not  striking. 
There  may  be  haemorrhages  in  the  muscles  which  have  been  the 
subject  of  the  spasms.  These  are  probably  due  to  mechanical 
causes.  Naturally  it  is  in  the  nervous  system  that  we  look  for 
the  most  important  lesions.  Here  there  is  ordinarily  a  general 
redness  of  the  grey  matter,  and  the  most  striking  feature  is  the 
occurrence  of  irregular  patches  of  slight  congestion  which  are  not 
limited  particularly  to  grey  or  white  matter,  or  to  any  tract  of 
the  latter.  These  patches  are  usually  best  marked  in  the  grey 
-matter  of  the  medulla  and  pons.  Microscopically  there  is  little 


378  TETANUS 

of  a  definite  nature  to  be  found.  There  is  congestion,  and  there 
may  be  minute  haemorrhages  in  the  areas  noted  by  the  naked 
eye.  The  ganglion  cells  may  show  appearances  which  have 
been  regarded  as  degenerative  in  nature,  and  similar  changes 
have  been  described  in  the  white  matter.  The  only  marked 
feature  is  thus  a  vascular  disturbance  in  the  central  nervous 
system,  with  a  possible  tendency  to  degeneration  in  its  specialised 
cells.  Both  of  these  conditions  are  probably  due  to  the  action 
of  the  toxins  of  the  bacillus.  In  the  case  of  the  cellular  degenera- 
tions the  cells  have  been  observed  to  return  to  the  normal  under 
the  curative  influence  of  the  antitoxins  (vide  infra).  In  the 
other  organs  of  the  body  there  are  no  constant  changes. 

We  have  said  that  the  general  distribution  of  pathogenic 
bacteria  throughout  the  body  is  probably  a  relative  phenomenon, 
and  that  bacteria  usually  found  locally  may  occur  generally  and 
vice  versa.  With  regard  to  the  tetanus  bacillus  it  is,  however, 
probably  the  case  that  very  rarely,  if  ever,  are  the  organisms 
found  anywhere  except  in  the  local  lesion. 

(b)  The  Artificially -produced  Disease. — The  disease  can  be  com- 
municated to  animals  by  any  of  the  usual  methods  of  inoculation, 
but  does  not  arise  in  animals  fed  with  bacilli,  whether  these 
contain  spores  or  not.  Kitasato  found  that  pure  cultures, 
injected  subcutaneously  or  intravenously,  caused  death  in  mice, 
rats,  guinea-pigs,  and  rabbits.  In  mice,  symptoms  appear  in  a 
day,  and  death  occurs  in  two  or  three  days,  after  inoculation 
with  a  loopful  of  a  bouillon  culture.  The  other  animals 
mentioned  require  larger  doses,  and  death  does  not  occur  so 
rapidly.  Usually  in  animals  injected  subcutaneously  the  spasms 
begin  in  the  limb  nearest  the  point  of  inoculation.  In  intra-. 
venous  inoculation  the  spasms  begin  in  the  extensor  muscles  of 
the  trunk,  as  is  the  case  in  the  natural  disease  in  man.  After 
death  there  is  found  slight  hyperaemia  without  pus  formation,  at 
the  seat  of  inoculation.  The  bacilli  diminish  in  number,  and 
may  be  absent  at  the  time  of  death.  The  organs  generally  show 
little  change. 

Kitasato  states  that  in  his  earlier  experiments  the  quantity  of 
culture  medium  injected  along  with  the  bacilli  already  contained 
enough  of  the  poisonous  bodies  formed  by  the  bacilli  to  cause 
death.  The  symptoms  came  on  sooner  than  by  the  improved 
method  mentioned  below,  and  were,  therefore,  due  to  the  toxins 
already  present.  In  his  subsequent  work,  therefore,  he  employed 
splinters  of  wood  soaked  in  cultures  in  which  spores  were  present, 
and  subsequently  subjected  for  one  hour  to  a  temperature  of 
80°  C.  The  latter  treatment  not  only  killed  all  the  bacilli,  but, 


TOXINS   OF   THE   TETANUS   BACILLUS         379 

as  we  shall  see,  was  sufficient  to  destroy  the  activity  of  the  toxins. 
When  such  splinters  are  introduced  subcutaneously,  death  results 
by  the  development  of  the  spores  which  they  carry.  In  this  way 
he  completed  the  proof  that  the  bacilli  by  themselves  can  form 
toxins  in  the  body  and  produce  the  disease.  Further,  if  a  small 
quantity  of  garden  earth  be  placed  under  the  skin  of  a  mouse, 
death  from  tetanus  takes  place  in  a  great  many  cases.  [Some- 
times, however,  in  such  circumstances  death  occurs  without 
tetanic  symptoms,  and  is  not  due  to  the  tetanus  bacillus  but  to 
the  bacillus  of  malignant  oedema,  which  also  is  of  common 
occurrence  in  the  soil  (vide  infra).]  By  such  experiments,  supple- 
mented by  the  culture  experiments  mentioned,  the  natural 
habitats  of  the  b.  tetani,  as  given  above,  have  become  known. 

The  Toxins  of  the  Tetanus  Bacillus. — The  tetanus  bacillus 
being  thus  accepted  as  the  cause  of  the  disease,  we  have  to 
consider  how  it  produces  its  pathogenic  effects. 

Almost  contemporaneously  with  the  work  on  diphtheria  was  the 
attempt  made  with  regard  to  tetanus  to  explain  the  general  symptoms 
by  supposing  that  the  bacillus  could  excrete  soluble  poisons.  The 
earlier  results  in  which  certain  bases,  tetanin  and  tetanotoxin,  were 
said  to  have  been  isolated,  have  only  a  historic  interest,  as  they  were 
obtained  by  faulty  methods.  In  1890  Brieger  and  Fraenkel  announced 
that  they  had  isolated  a  toxalbumin  from  tetanus  cultures,  and  this  body 
was  independently  discovered  by  Faber  in  the  same  year.  Brieger  and 
Fraenkel's  body  consisted  practically  of  an  alcoholic  precipitate  from 
filtered  cultures  in  bouillon,  and  was  undoubtedly  toxic.  Within  recent 
years  such  attempts  to  isolate  tetanus  toxins  in  a  pure  condition  have 
practically  been  abandoned,  and  attention  has  been  turned  to  the 
investigation  of  the  physiological  effects  either  of  the  crude  toxin 
present  in  filtered  bouillon  cultures,  or  of  the  precipitate  produced  from 
the  same  by  ammonium  sulphate  (cf.  p.  167). 

The  toxic  properties  of  bacterium-free  filtrates  of  pure  cultures 
of  the  b.  tetani  were  investigated  in  1891  by  Kitasato.  This 
observer  found  that  when  the  filtrate,  in  certain  doses,  was 
injected  subcutaneously  or  intravenously  into  mice,  tetanic  spasms 
developed,  first  in  muscles  contiguous  to  the  site  of  inoculation, 
and  later  all  over  the  body.  Death  resulted.  He  found  that 
guinea-pigs  were  more  susceptible  than  mice,  and  rabbits  less  so. 
In  order  that  a  strongly  toxic  bouillon  be  produced,  it  must 
originally  have  been  either  neutral  or  slightly  alkaline.  Kitasato 
further  found  that  the  toxin  was  easily  injured  by  heat.  Exposure 
for  a  few  minutes  at  65°  C.  destroyed  it.  It  was  also  destroyed 
by  twenty  minutes'  exposure  at  60°  C.,  and  by  one  and  a  half 
hours'  at  55°  C.  Drying  had  no  effect.  It  was,  however, 
destroyed  by  various  chemicals  such  as  pyrogallol  and  also  by 


380  TETANUS 

sunlight.  Behring  has  more  recently  pointed  out  that  after  the 
nitration  of  cultures  containing  toxin,  the  latter  may  very  rapidly 
'lose  its  power,  and  in  a  few  days  may  only  possess  y-g-g-th  of  its 
original  toxicity.  This  he  attributes  to  such  factors  as  temperature 
and  light,  and  especially  to  the  action  of  oxygen.  The  effect 
of  these  agents  on  the  crude  toxin  is  undoubtedly  to  cause  a 
degeneration  of  the  true  toxin  into  a  series  of  toxoids  similar  to 
those  produced  in  the  case  of  diphtheria  toxin,  and  it  is  also 
true  here  that  the  toxoids  while  losing  their  toxicity  may  still 
retain  their  power  of  producing  immunity  against  the  potent 
toxin.  Further,  altogether  apart  from  the  occurrence  side  by 
side  in  the  crude  toxin  of  strong  and  weak  poisons,  it  has  been 
shown  that  such  crude  toxin  contains  toxic  substances  of  probably 
quite  a  different  nature.  Ehrlich  has  shown  that  besides  the  pre- 
dominant spasm-producing  toxin  (called  by  him  tetanospasmin), 
there  exists  in  crude  toxin  a  poison  capable  of  producing  the 
solution  of  certain  red  blood  corpuscles.  This  hsemolytic  agent 
he  calls  tetanolysin.  It  does  not  occur  in  all  samples  of  crude 
tetanus  toxin,  nor  is  it  found  when  a  bouillon  culture  of  the 
bacillus  is  filtered  through  porcelain.  To  obtain  it  the  fresh 
culture  must  be  treated  by  ammonium  sulphate,  as  described  in 
the  method  of  obtaining  concentrated  toxins  (p.  167).  This 
substance  also  has  the  power  of  originating  an  antitoxin  so  that 
certain  antitetanic  sera  can  protect  red  blood  corpuscles  against 
its  action.  Madsen,  studying  the  interactions  of  this  anti- 
tetanolysin  with  the  tetanolysin,  has  shown  that  the  phenomena 
can  be  demonstrated  similar  to  those  noted  by  Ehrlich  as 
occurring  with  diphtheria  toxin,  and  which  he  interpreted  as 
indicating  the  presence  of  degenerated  toxins  (toxoids)  in  the 
crude  poison.  With  tetanus  as  with  diphtheria  toxin  the  action 
of  an  acid  is  to  cause  an  apparent  disappearance  of  toxicity,  but 
if  before  a  certain  time  has  elapsed  the  acid  be  neutralised  by 
alkali,  then  a  degree  of  the  toxicity  returns. 

As  with  other  members  of  the  group,  nothing  is  known  of  the 
nature  of  tetanus  toxin.  Uschinsky  has  found  that  the  tetanus 
bacillus  can  produce  its  toxin  when  growing  in  a  fluid  containing 
no  proteid  matter.  The  toxin  may  thus  be  formed  independently 
of  the  breaking  up  of  the  proteids  on  which  the  bacillus  may  be 
living,  though  the  latter  no  doubt  has  a  digestive  action  on 
these.  The  liquefaction  (i.e.  probable  peptonisation)  of  gelatin 
cultures  advances  pari  passu  with  the  development  of  toxins, 
and  filtered  bacterium-free  cultures  will  still  liquefy  gelatin.  It 
is  probable  that  there  is  an  independent  peptic  ferment  which 
will,  of  course,  also  pass  through  a  filter.  For  if  equal  portions 


TOXINS   OF   THE   TETANUS   BACILLUS         381 

of  the  filtered  culture  be  left  in  contact  with  equal  portions  of 
gelatin  for  various  lengths  of  time,  there  is  no  increase  of  toxicity 
in  those  kept  longest.  There  is  thus  no  fresh  development  of 
toxin  during  the  advancing  liquefaction  of  the  gelatin.  Thus 
peptic  digestion  and  toxin  formation  are  apparently  due  to 
different  vital  processes  on  the  part  of  the  tetanus  bacillus. 

Whatever  the  nature  of  the  toxin  is,  it  is  undoubtedly  one 
of  the  most  powerful  poisons  known.  Even  with  a  probably 
impure  toxalbumin  Brieger  found  that  the  fatal  dose  for  a 
mouse  was  '0005  of  a  milligramme.  If  the  susceptibility  of 
man  be  the  same  as  that  of  a  mouse,  the  fatal  dose  for  an  average 
adult  would  have  been  '23  of  a  milligramme,  or  about  ^^^ths 
of  a  grain.  Animals  differ  very  much  in  their  susceptibilities 
to  the  action  of  tetanus  toxin.  According  to  v.  Lingelsheim,  if 
the  minimal  lethal  dose  per  gramme  weight  for  a  horse  be  taken 
as  unity,  that  for  the  guinea-pig  would  be  6  times  the  amount, 
the  mouse  12,  the  goat  24,  the  dog  about  500,  the  rabbit  1800, 
the  cat  6000,  the  goose  12,000,  the  pigeon  48,000,  and  the 
hen  360,000. 

A  striking  feature  of  the  action  of  tetanus  toxin  is  the 
occurrence  of  a  definite  incubation  period  between  the  introduc- 
tion of  the  toxin  into  an  animal's  body  and  the  appearance  of 
symptoms.  The  incubation  period  varies  according  to  the  species 
of  animal  employed,  and  the  path  of  infection.  In  the  guinea- 
pig  it  is  from  thirteen  to  eighteen  hours,  in  the  horse  five  days, 
and  the  incubation  is  shorter  when  the  poison  is  introduced  into 
a  vein  than  when  injected  subcutaneously.  In  man  the  period 
between  the  receiving  of  an  injury  and  the  appearance  of  tetanic 
symptoms  is  from  two  to  fourteen  days. 

With  regard  to  the  action  of  the  toxin,  it  has  been  shown  to 
have  no  effect  on  the  sensory  or  motor  endings  of  the  nerves. 
It  acts  solely  as  an  exciter  of  the  reflex  excitability  of  the  motor 
cells  in  the  spinal  cord.  The  motor  cells  in  the  pons  and 
medulla  are  also  affected,  and  to  a  much  greater  degree  than 
those  in  the  cerebral  cortex.  When  injected  subcutaneously 
the  toxin  is  absorbed  into  the  nerves,  and  thence  finds  its  way 
to  that  part  of  the  spinal  cord  from  which  these  nerves  spring. 
This  explains  the  fact  that  in  some  animals  the  tetanic  spasms 
appear  first  in  the  muscles  of  the  part  in  which  the  inoculation 
has  taken  place.  This  is  not  the  case  with  man,  in  whom  usually 
the  first  symptoms  appear  in  the  neck.  In  artificial  injection  of 
toxin  part  finds  its  way  into  the  blood  stream,  and  if  infected 
animals  be  killed  during  the  incubation  period  there  is  often 
evidence  of  toxin  in  the  blood  and  solid  organs.  In  the  guinea- 


382  TETANUS 

pig  there  is  little  doubt  that  tetanus  toxin  has  an  affinity  solely 
for  the  nervous  system.  In  other  animals,  such  as  the  rabbit, 
an  affinity  may  exist  in  other  organs,  and  the  fixation  of  the 
poison  in  such  situations  may  give  rise  to  no  recognisable 
symptoms.  In  such  an  animal  as  the  alligator,  it  is  possible 
that  while  some  of  its  organs  have  an  affinity  for  tetanus  toxin 
its  nervous  system  has  none.  These  facts  are  of  great  scientific 
interest,  and  a  possible  explanation  of  them  will  be  discussed  in 
the  chapter  on  Immunity.  If  tetanus  toxin  be  introduced  into 
the  stomach  or  intestine,  it  is  not  absorbed,  but  to  a  large  extent 
passes  through  the  intestine  unchanged.  Evidence  that  any 
destruction  takes  place  is  wanting. 

Within  recent  years  some  important  light  has  been  shed  on 
the  mode  of  action  of  tetanus  toxin.  Marie  and  Morax  studied 
the  path  of  absorption  when  the  toxin  was  injected  into  the 
muscles  of  the  hind  limb.  The  sciatic  nerve  in  a  rabbit  was  cut 
near  the  spinal  cord  and  toxin  introduced  into  the  muscles  of  the 
same  side ;  after  some  hours  the  nerve  was  excised  and  introduced 
into  a  mouse — the  animal  died  of  tetanus.  But  if  the  nerve  were 
cut  near  the  muscles  and  the  same  procedure  adopted,  the  mouse 
did  not  contract  the  disease,  though  no  doubt  the  cut  nerve  had 
been  surrounded  by  lymph  containing  toxin.  If  the  same 
experiment  were  performed  and  an  excess  of  toxin  injected  into 
the  other  limb,  still  only  the  nerve  which  was  left  in  connection 
with  the  muscle  showed  evidence  of  containing  toxin.  From 
this  it  was  deduced  that  the  toxin  was  absorbed  by  the  end- 
plates  in  the  muscle  and  not  from  the  lymphatics  surrounding 
the  nerve.  It  was  further  shown  that  a  nerve  in  the  process  of 
degeneration  following  section  did  not  absorb  toxin  after  the 
manner  of  a  normal  nerve.  By  a  similar  method  it  was  shown 
that  the  absorption  by  the  nerve  was  fairly  rapid,  as  one  hour 
after  injection  the  toxin  was  present  in  it,  and  from  other 
experiments  the  view  was  put  forth  that  the  toxin  was  centripetal 
in  its  flow  and  did  not  pass  centrifugally  in  a  nerve  to  which  it 
artificially  gained  access.  Further  observations  have  been  made 
on  this  subject  by  Meyer  and  Ransom.  These  observers  found 
evidence  that  toxin  is  only  absorbed  by  the  motor  filaments  of  a 
nerve,  for  while  tetanus  could  be  produced  by  injection  into  a 
mixed  nerve  like  the  sciatic  the  introduction  of  a  lethal  dose  into 
such  a  sensory  nerve  as  the  infra-orbital  was  not  followed  by 
disease  symptoms.  If  a  small  dose  of  toxin  be  injected  into  the 
sciatic  nerve  it  reaches  the  corresponding  motor  cells  of  the  cord, 
and  a  local  tetanus  of  the  muscles  supplied  by  the  nerve  results. 
With  a  larger  dose  the  poison  passes  across  the  commissure  to 


TOXINS   OF   THE   TETANUS   BACILLUS         383 

the  corresponding  cells  of  the  other  side,  and  if  still  further 
excess  is  present  it  passes  up  the  cord  to  higher  centres.  The 
affection  of  such  higher  centres  can  be  prevented  by  section  of 
the  cord.  Meyer  and  Ransom  hold  that  when  toxin  is  injected 
subcutaneously  or  intravenously,  it  only  acts  by  being  absorbed 
by  the  end-plates  in  muscles  and  thence  passes  to  the  cord,  and 
they  consider  that  the  incubation  period  is  to  be  explained  by  the 
time  taken  for  this  extended  passage  to  occur.  In  this  connection 
they  point  out  that  it  is  in  the  larger  animals,  where  the  nerve 
path  is  longest,  that  the  incubation  period  is  also  long.  Like 
Marie  and  Morax,  they  believe  that  absorption  of  toxin  by  its 
bathing  the  lateral  aspects  of  uninjured  nervous  structures  does 
not  occur,  and  in  support  of  this  they  bring  forward  the 
observation  that  when  intravenous  injection  is  practised,  the 
occurrence  of  tetanus  in  a  part  of  the  body  can  be  precipitated  by 
such  a  slight  injury  as  may  be  caused  by  the  injection  of  a  drop 
of  normal  saline  into  the  corresponding  part  of  the  cord.  With 
regard  to  the  action  of  tetanus  toxin,  Meyer  and  Ransom  believe 
that  there  is  a  double  effect  on  the  nerve  cells — first,  an  exaggera- 
tion of  the  normal  tonus,  which  accounts  for  the  continuous 
stiffness  of  the  muscles,  and  secondly,  an  increase  in  reflex 
irritability,  which  is  a  prominent  factor  in  the  recurring  spasms. 
While  no  absorption  of  toxin  takes  place  by  sensory  filaments, 
they  have  found  evidence  of  sensibility  of  the  sensory  apparatus 
in  the  occurrence  of  what  they  call  tetanus  dolorosus.  This  is  a 
great  hyperaesthesia  and  a  paroxysmal  hyperalgesia  which  can  be 
caused  by  injecting  toxin  into  the  spinal  cord  or  into  a  sensory 
root  on  the  spinal  side  of  the  posterior  root  ganglion.  These 
symptoms  are  unaccompanied  by  motor  spasms,  but  the  animal 
may  die  from  exhaustion.  The  same  observers  have  also  made 
interesting  observations  on  the  action  of  antitoxin.  They  found 
that  the  injection  of  this  substance  into  the  course  of  a  mixed 
nerve  could  prevent  toxin  from  passing  up  to  the  cord,  but  that  if 
antitoxin  were  injected  even  in  great  excess  intravenously,  and  a 
short  time  thereafter  toxin  were  introduced  into  a  nerve,  the 
death  of  the  animal  was  not  prevented.  This  they  attribute 
to  the  fact  that  antitoxin  can  only  neutralise  the  toxin  which  is 
still  circulating  in  the  blood.  This  is  a  very  far-reaching 
conclusion,  as  it  throws  doubt  on  what  has  been  held  to  be  a 
possibility,  namely,  that  toxin  can  be  actually  detached  from 
cells  in  which  it  is  already  anchored.  But  a  still  more 
significant  observation  was  made,  for  in  one  case  of  an  animal 
actively  immunised  against  tetanus,  and  which  contained  in  its 
serum  a  considerable  quantity  of  antitoxin,  the  injection  of  toxin 


384  TETANUS 

into  the  sciatic  nerve  was  followed  by  tetanus.  This  would 
appear  to  militate  against  Ehrlich's  position  that  antitoxin  is 
manufactured  in  the  cells  which  are  sensitive  to  the  toxin  (see 
Immunity). 

Reference  may  here  be  made  to  the  effects  of  injecting  tetanus 
toxin  into  the  brain  itself,  as  investigated  by  Roux  and  Borrel. 
It  was  found  that  the  ordinary  type  of  the  disease  was  not 
produced,  but  what  these  observers  called  "cerebral  tetanus." 
This  consisted  of  general  unrest,  symptoms  of  a  psychic 
character  (apparent  hallucinations, fear,  etc.),  and  epileptiform  con- 
vulsions. Death  occurred  in  from  twelve  to  twenty  hours  without 
any  true  tetanic  spasms.  In  this  manifestation  of  tetanus  the 
incubation  period  was  much  shorter  than  with  subcutaneous 
injection,  and  the  fatal  dose  was  one  twenty-fifth  of  the  minimal 
subcutaneous  dose.  Further,  the  injection  of  antitoxin  forty-eight 
to  ninety-six  hours  previously  did  not  prevent  an  animal  from  suc- 
cumbing to  the  intracerebral  inoculation.  In  the  light  of  what  has 
been  already  said  these  results  would  seem  to  indicate  a  special 
effect  of  the  toxin  when  brought  into  direct  contact  with  the 
protoplasm  of  the  brain  cells. 

We  have  seen,  that  unless  suitable  precautions  are  adopted,  in 
experimental  tetanus  in  animals  death  results  not  from  inocula- 
tion but  from  an  intoxication  with  toxin  previously  existent  in 
the  fluid  in  which  the  bacilli  have  been  growing.  According  to 
Vaillard,  if  spores  rendered  toxin-free,  by  being  kept  for  a 
sufficient  time  at  80°  C.,  are  injected  into  an  animal,  death  does 
not  take  place.  It  was  found,  however,  that  such  spores  can  be 
rendered  pathogenic  by  injecting  along  with  them  such  chemicals 
as  lactic  acid,  by  injuring  the  seat  of  inoculation  so  as  to  cause 
effusion  of  blood,  by  fracturing  an  adjacent  bone,  by  introducing 
a  mechanical  irritant  such  as  soil  or  a  splinter  of  wood  (as  in 
Kitasato's  experiments),  or  by  the  simultaneous  injection  of 
other  tjacteria  such  as  the  staphylococcus  pyogenes  aureus.  These 
facts,  especially  the  last,  throw  great  light  on  the  disease  as  it 
occurs  naturally,  for  tetanus  results  especially  from  wounds 
which  have  been  accidentally  subjected  to  conditions  such  as 
those  enumerated.  Kitasato  now  holds  that  in  the  natural 
infection  in  man,  along  with  tetanus  spores,  the  presence  of 
foreign  material  or  of  other  bacteria  is  necessary.  Spores  alone 
or  tetanus  bacilli  without  spores  die  in  the  tissues,  and  tetanus 
does  not  result. 

Immunity  against  Tetanus. — Antitetanic  Serum. — The  arti- 
ficial immunisation  of  animals  against  tetanus  has  received  much 
attention.  The  most  complete  study  of  the  question  is  found 


IMMUNITY   AGAINST   TETANUS  385 

in  the  work  of  Behring  and  Kitasato  in  Germany,  and  of  Tizzoni 
and  Cattani  in  Italy.  The  former  observers  found  that  such  an 
immunity  could  be  conferred  by  the  injection  of  very  small  and 
progressively  increasing  doses  of  the  tetanus  toxin.  The  degree 
of  immunity  attained,  however,  was  not  high.  Subsequent 
work  has  shown  that  the  less  rich  a  crude  toxin  is  in  modifica- 
tions of  the  true  toxin  the  less  useful  it  is  for  immunisation 
procedures.  In  fact  it  is  doubtful  if  small  animals  can  be 
immunised  at  all  by  fresh  filtrates.  In  some  cases  it  has  been 
found  that  the  injection  of  non-lethal  doses  instead  of  commenc- 
ing an  immunity  actually  increases  the  susceptibility  of  the 
animal.  More  successful  are  the  methods  of  accompanying  the 
early  injections  of  crude  toxin  with  the  subcutaneous  introduction 
of  small  doses  of  iodine  terchloride,  or  of  using  toxin  which  has  been 
acted  on  with  iodine  terchloride  or  with  iodine  itself.  Tizzoni 
and  Cattani  have  also  used  the  method  of  administering  pro- 
gressively increasing  doses  of  living  cultures  attenuated  in 
various  ways,  e.g.  by  heat.  By  any  of  these  methods  susceptible 
animals  can  be  made  to  acquire  great  immunity,  not  only 
against  many  times  the  fatal  dose  of  tetanic  toxin,  but  also 
against  injections  of  the  living  bacilli.  The  degree  of  immunisa- 
tion acquired  by  an  animal  remains  in  existence  for  a  very  long 
time.  Not  only  so,  but  when  a  high  degree  of  immunity  has 
been  produced  by  prolonged  treatment,  it  is  found  that  the 
serum  of  immune  animals  possesses  the  capacity,  when  injected 
into  animals  susceptible  to  the  disease,  of  protecting  them 
against  a  subsequent  infection  with  a  fatal  dose  of  tetanus 
bacilli  or  toxin.  Further,  if  injected  subsequently  to  such 
infection,  the  serum  can  in  certain  cases  prevent  a  fatal  result, 
even  when  symptoms  have  begun  to  appear.  The  degree  of 
success  attained  depends,  however,  on  the  shortness  of  the  time 
which  has  elapsed  between  the  infection  with  the  bacilli  or  toxin 
and  the  injection  of  the  serum.  In  animals  where  symptoms 
have  fully  manifested  themselves  only  a  small  proportion  of 
cases  can  be  saved.  As  with  other  antitoxins  there  is  no 
evidence  that  the  antitetanic  serum  has  any  detrimental  effect 
on  the  bacilli.  It  only  neutralises  the  effects  of  the  toxin. 
The  standardisation  of  the  antitetanic  serum  is  of  the  highest 
importance.  Behring  recommends  that  for  protecting  animals 
a  serum  should  be  obtained  of  which  one  gramme  will  protect 
1,000,000  grammes  weight  of  mice  against  the  minimum  fatal 
dose  of  the  bacillus  or  toxin.  A  mouse  weighing  twenty 
grammes  would  thus  require  "00002  gramme  of  the  serum  to 
protect  it  against  the  minimum  lethal  dose.  In  the  injection 
25 


386  TETANUS 

of  such  a  serum  subsequent  to  infection,  if  symptoms  have 
begun  to  appear,  1000  times  this  dose  would  be  necessary;  a 
few  hours  later  10,000  times,  and  so  on. 

As  the  result  of  his  experiments,  Behring  aimed  at  obtaining 
a  curative  effect  in  the  natural  disease  occurring  in  man.  For 
this  purpose,  as  for  his  later  laboratory  experiments,  he  obtained 
serum  by  the  immunisation  of  such  large  animals  as  the  horse, 
the  sheep,  and  the  goat,  by  the  injection  of  toxin  accompanied 
at  first  with  the  injection  of  iodine  terchloride.  It  was  found 
that  the  greater  the  degree  of  the  natural  susceptibility  of  an 
animal  to  tetanus,  the  easier  was  it  to  obtain  a  serum  of  a  high 
antitetanic  potency.  The  horse  was,  therefore,  the  most 
suitable  animal.  If  now  we  take  for  granted  that  the  relative 
susceptibility  of  man  and  the  mouse  towards  tetanus  are  nearly 
equal,  a  man  weighing  100  kilogrm.  would  require  '1  grm.  of 
the  serum  mentioned  above  to  protect  him  from  inoculation 
with  the  minimum  lethal  dose  of  bacilli  or  toxin.  If  symptoms 
had  begun  to  appear,  100  c.c.  at  once  would  be  necessary,  and 
as  the  injection  of  such  a  quantity  might  be  inconvenient, 
Behring  recommended  that  for  man  a  more  powerful  serum 
should  be  obtained,  viz.,  a  serum  of  which  one  gramme  would 
protect  100,000,000  grammes  weight  of  mice.1  The  potency  is 
maintained  for  several  months  if  precautions  are  taken  to 
avoid  putrefaction,  exposure  to  bright  light,  etc.  To  this  end 
*5  per  cent  carbolic  acid  is  usually  added,  and  the  serum  is 
kept  in  the  dark.  In  a  case  of  tetanus  in  man,  100  c.c.  of 
such  a  serum  should  be  injected  within  twenty-four  hours  in 
five  doses,  each  at  a  different  part  of  the  body,  and  this 
followed  up  by  further  injections  if  no  improvement  takes 
place.  Intravenous  injection  of  the  antitoxin  has  also  been 
practised,  and,  in  cases  which  we  have  seen  treated  in  this  way, 
has  seemed  to  give  better  results  than  those  obtained  by  the 
subcutaneous  method.  The  serum  is  warmed  to  the  body 
temperature  and  slowly  introduced  into  a  vein  in  the  arm,  the 
pulse  and  respiration  being  carefully  watched  during  the 
proceeding.  Ten  to  twenty  c.c.  can  be  injected  every  few  hours, 
and  in  all  100  c.c.  should  be  given  in  as  short  a  time  as  possible. 
Henderson  Smith  has  shown  that  when  antitoxins  to  toxins  of 
the  tetanus  group  are  injected  intravenously  a  high  concentration 
in  the  body  fluid  is  maintained  for  some  time  and  the  op- 
portunity for  neutralisation  of  toxin  is  thus  great.  He  suggests 

1  The  antitetanic  serum  sent  out  by  the  Pasteur  Institute  in  Paris  has  a 
strength  of  1  :  1,000,000,000.  Of  this  it  is  recommended  that  50  to  100  c.c. 
should  be  injected  in  one  or  two  doses. 


IMMUNITY  AGAINST   TETANUS  387 

that  both  intravenous  and  subcutaneous  injections  should  be 
simultaneously  practised.  The  former  gives  the  quickly  attained 
concentration  which  is  desirable,  and  when  the  antitoxin  injected 
intravenously  is  beginning  to  be  eliminated  that  introduced 
hypodermically  comes  into  the  circulation  and  the  concentration 
is  maintained.  The  antitoxin  has  also  been  introduced  intra- 
cerebrally,  very  slow  injection  into  the  brain  substance  being 
practised,  but  no  better  results  have  been  obtained  than  by  the 
subcutaneous  method. 

Many  cases  of  human  tetanus  have  been  thus  treated,  but 
the  improvement  in  the  death-rate  has  not  been  nearly  so 
marked  as  that  which  has  occurred  in  diphtheria  under  similar 
circumstances.  As  in  the  case  of  diphtheria,  however,  the 
results  would  probably  be  better  if  more  attention  were  paid 
to  the  dosage  of  the  serum.  The  great  difficulty  is  that,  as  a 
matter  of  fact,  we  have  not  the  opportunity  of  recognising  the 
presence  of  the  tetanus  bacilli  till  they  have  begun  to  manifest 
their  gravest  effects.  In  diphtheria  we  have  a  well-marked 
clinical  feature  which  draws  attention  to  the  probable  presence 
of  the  bacilli — a  presence  which  can  be  readily  proved, — and 
the  curative  agent  can  thus  be  early  applied.  In  tetanus  the 
wound  in  which  the  bacilli  exist  may  be,  as  we  have  seen,  of 
the  most  trifling  character,  and  even  when  a  well-marked 
wound  exists,  the  search  for  the  bacilli  may  be  a  matter  of 
difficulty.  Still  it  might  be  well,  when  practicable,  that  every 
ragged,  unhealthy-looking  wound,  especially  when  contaminated 
with  soil,  should,  as  a  matter  of  routine,  be  examined 
bacteriologically.  In  such  cases,  undoubtedly,  from  time  to 
time  cases  of  tetanus  would  be  detected  early,  and  their  treat- 
ment could  be  undertaken  with  more  hope  of  success  than  at 
present.  However,  in  the  existing  state  of  matters,  whenever 
the  first  symptoms  of  tetanus  appear,  large  doses,  such  as  those 
above  indicated,  of  a  serum  whose  strength  is  known,  should  be 
at  once  administered.  In  giving  a  prognosis  as  to  the  probable 
result,  the  two  clinical  observations  on  which,  according  to 
Behring,  chief  reliance  ought  to  be  placed,  are  the  presence  or 
absence  of  interference  with  respiration,  and  the  rapidity  with 
which  the  groups  of  muscles  usually  affected  are  attacked.  If 
dyspnoea  or  irregularity  in  respiration  comes  on  soon,  and  if  group 
after  group  of  muscles  is  quickly  involved,  then  the  outlook  is 
extremely  grave.  In  addition  to  these  points  the  duration  of  the 
incubation  period  is  of  high  importance  in  forming  a  prognosis. 
The  shorter  the  time  between  the  infliction  of  a  wound  and  the 
appearance  of  symptoms  the  graver  is  the  outlook. 


388  TETANUS 

The  theory  as  to  the  nature  of  antitoxic  action  will  be 
discussed  later  in  the  chapter  on  Immunity. 

Methods  of  Examination  in  a  case  of  Tetanus. — The 
routine  bacteriological  procedure  in  a  case  presenting  the  clinical 
features  of  tetanus  ought  to  be  as  follows  :— 

(a)  Microscopic. — Though  tetanus  is  not  a  disease  in  which 
the  discovery  of  the  bacilli  is  easy,  still  microscopic  examination 
should  be  undertaken  in  every  case.     From   every  wound  or 
abrasion  from  which  sufficient  discharge  can  be  obtained,  film 
preparations  ought  to  be  made  and  stained  with  any  of  the 
ordinary  combinations,  e.g.  carbol-fuchsin  diluted  with  five  parts 
of  water.      Drumstick-shaped    spore-bearing   bacilli  are   to  be 
looked   for.     The   presence   of   such,   having   characters   corre- 
sponding to  those  of  the  tetanus  bacilli,  though  not  absolutely 
conclusive  proof  of  identification,  is  yet  sufficient  for  all  practical 
purposes.     If  only  bacilli  without  spores  resembling  the  tetanus 
bacilli  are  seen,  then  the  identification  can  only  be  provisional. 

The  microscopic  examination  of  wounds  contaminated  by  soil, 
etc.,  may,  as  we  have  said,  in  some  cases  lead  to  the  anticipation 
that  tetanus  will  probably  result. 

(b)  Cultivation. — The  methods  to  be  employed  in  isolating 
the  tetanus  bacilli  have  already  been  described  (p.  373).     It 
may  be  added,  however,  that    if    the  characteristic  forms  are 
not  seen  on  microscopic  examination  of  the  material  from  the 
wound,  they  may  often  be  found  by  inoculating  a  deep  tube  of 
one  of  the  glucose  media  with  such  material,  and  incubating  for 
forty-eight  hours  at  37°  C.     At  the  end  of  this  period,  spore- 
bearing  tetanus  bacilli  may  be  detected  microscopically,  though 
of  course  mixed  with  other  organisms. 

(c)  Inoculation.-  -Mice  and  guinea-pigs  are  the  most  suitable 
animals.     Inoculation  with  the  material  from  a  wound  should 
be  made  subcutaneously.     A  loopful  of  the  discharge  introduced 
at  the  root  of  the  tail  in  a  mouse  will  soon   give  rise  to  the 
characteristic  symptoms,  if  tetanus  bacilli  are  present. 

MALIGNANT  (EDEMA  (Septicemie  de  Pasteur). 

The  organism  now  usually  known  as  the  bacillus  of  malignant 
oedema  is  the  same  as  that  first  discovered  by  Pasteur  and 
named  vibrion  septique.  He  described  its  characters,  distin- 
guishing it  from  the  anthrax  bacillus  which  it  somewhat 
resembles  morphologically,  and  also  the  lesions  produced  by  it. 
He  found  that  it  grew  only  in  anaerobic  conditions,  but  was 
able  to  cultivate  it  merely  in  an  impure  state.  It  was  more 


MALIGNANT   (EDEMA 


389 


fully  studied  by  Koch,  who  called  it  the  bacillus  of  malignant 
oedema,  and  pointed  out  that  the  disease  produced  by  it  is  not 
really  of  the  nature  of  a  septicaemia,  as  immediately  after  death 
the  blood  is  practically  free  from  the  bacilli. 

"Malignant  oedema"  in  the  human  subject  is  usually 
described  as  a  spreading  inflammatory  oedema  attended  with 
emphysema,  and  ultimately  followed  by  gangrene  of  the  skin 


FIG.  129. — Film  preparation  from  the  affected  tissues  in  a  case  of  malignant 
.    oedema  in  the  human  subject,  showing  the  spore-bearing  bacilli. 
Gentian- violet.      x  1000. 

and  subjacent  parts.  In  many  cases  of  this  nature  the  bacillus 
of  malignant  oedema  is  present,  associated  with  other  organisms 
which  aid  its  spread,  whilst  in  others  it  may  be  absznt.  One  of 
us  has,  however,  observed  a  case  in  which  the  bacillus  was 
present  in  pure  condition.  Here  there  occurred  intense  oedema 
with  swelling  and  induration  of  the  Jtissues,  and  the  formation 
of  vesicles  on  the  skin.  Those  changes  were  attended  with  a 
reddish  discoloration,  afterwards  becoming  livid.  Emphysema 
was  not  recognisable  until  the  limb  was  incised,  when  it  was 
detected,  though  in  small  degree.  Further,  the  tissues  had  a 


390 


MALIGNANT   (EDEMA 


peculiar  heavy,  but  not  putrid,  odour.  The  bacillus,  which  was 
obtained  in  pure  culture,  was  present  in  enormous  numbers  in 
the  affected  tissues,  attended  by  cellular  necrosis,  serous 
exudation,  and  at  places  much  leucocytic  emigration.  The 
picture,  in  short,  corresponded  with  that  seen  on  inoculating  a 
guinea-pig  with  a  pure  culture.  The  term  "malignant  oedema" 
should  be  limited  in  its  application  to  cases  in  which  the 
bacillus  in  question  is  present.  In  most  of  these  there  is  a 
mixed  infection ;  in  some  this  bacillus  may  be  present  alone. 
This  bacillus  has  a  very  widespread  distribution  in  nature, 

being  present  in  garden 
soil,  dung,  and  various 
putrefying  animal  fluids ; 
and  it  is  by  contamination 
of  lacerated  wounds  by  such 
substances  that  the  disease 
is  usually  set  up  in  the 
human  subject.  Malignant 
oedema  can  be  readily  pro- 
duced by  inoculating  sus- 
ceptible animals,  such  as 
guinea-pigs,  with  garden 
soil.  The  bacillus  is  also 
of  ten  present  in  the  intestine 
of  man  and  animals,  and  has 
been  described  as  being 
FIG.  130. — Bacillus  of  malignant  oedema,  present  in  some  gangrenous 


showing   spores.       From    a   culture    in 
glucose  agar,  incubated  for  three  days 
at  37°  C. 
Stained  with  weak  ca'rbol-fuchsin.      x  1000. 


conditions  originating  in 
connection  with  the  in- 
testine in  the  human  sub- 
ject. 

Microscopical  Characters.— The  bacillus  of  malignant  oedema 
is  a  comparatively  large  organism,  being  slightly  less  than  1  /A 
in  thickness,  that  is,  thinner  than  the  anthrax  bacillus.  It 
occurs  in  the  form  of  single  rods  3  /x  to  10  ^  in  length,  but  both 
in  the  tissues  and  in  cultures  in  fluids  it  frequently  grows  out 
into  long  filaments,  which  may  be  uniform  throughout  or 
segmented  at  irregular  intervals.  In  cultures  on  solid  media  it 
chiefly  occurs  in  the  form  of  shorter  rods  with  somewhat  rounded 
ends.  The  rods  are  motile,  possessing  several  laterally  placed 
flagella,  but  in  a  given  specimen,  as  a  rule,  only  a  few  bacilli 
show  active  movement.  Under  suitable  conditions  they  form 
spores  which  are  usually  near  the  centre  of  the  rods  and  have  an 
oval  shape,  their  thickness  somewhat  exceeding  that  of  the 


CHARACTER   OF   CULTURES 


391 


bacillus  (Figs.  129,  130).  The  bacillus  can  be  readily  stained 
by  any  of  the  basic  aniline  stains,  but  loses  the  colour  in  Gram's 
method,  in  this  way  differing  from  the  anthrax  bacillus. 

Characters  of  Cultures. — This  organism  grows  readily  at 
ordinary  temperature,  but  only  under  anaerobic  conditions.  In 
a  puncture  culture  in  a  deep  tube  of  glucose  gelatin,  the  growth 


n 


A  B  C 

Fio.  13L — Stab  cultures  in  agar,  five  days'  growth  at  37°  C.     Natural  size. 

A.  Tetanus  bacillus.     B.  Bacillus  of  malignant  oedema.     C.  Bacillus  of 
quarter-evil  (Rauschbrand). 

appears  as  a  whitish  line  giving  off  minute  short  processes,  the 
growth,  of"  course,  not  reaching  the  surface  of  the  medium. 
Soon  liquefaction  occurs,  and  a  long  fluid  funnel  is  formed,  with 
turbid  contents  and  flocculent  masses  of  growth  at  the  bottom. 
At  the  same  time  bubbles  of  gas  are  given  off,  which  may  split 
up  the  gelatin.  The  colonies  in  gelatin  plates  under  anaerobic 
conditions  appear  first  as  small  whitish  points  which  under  the 
microscope  show  a  radiating  appearance  at  the  periphery, 
resembling  the  colonies  of  the  bacillus  subtilis.  Soon,  however, 


392  MALIGNANT   (EDEMA 

liquefaction  occurs  around  the  colonies,  and  spheres  with  turbid 
contents  result ;  gas  is  developed  around  the  colonies. 

In  deep  tubes  of  glucose  agar  at  37°  C.,  growth  is  extremely 
rapid.  Along  the  line  of  puncture,  growth  appears  as  a  some- 
what broad  white  line  with  short  lateral  projections  here  and 
there  (Fig.  131,  B).  Here  also  gas  may  be  formed,  but  this  is 
most  marked  in  a  shake  culture,  in  which  the  medium  becomes 
cracked  in  various  directions,  and  may  be  pushed  upwards  so 
high  as  to  displace  the  cotton-wool  plug.  The  cultures  possess 
a  peculiar  heavy,  though  not  putrid,  odour. 

Spore  formation  occurs  above  20°  C.,  and  is  usually, well 
seen  within  forty-eight  hours  at  37°  C.  The  spores  have  the 
usual  high  powers  of  resistance,  and  may  be  kept  for  months  in 
the  dried  condition  without  being  killed. 

Experimental  Inoculation. — A  considerable  number  of  animals 
— the  guinea-pig,  rabbit,  sheep,  and  goat,  for  example — are 
susceptible  to  inoculation  with  this  organism.  The  ox  is  said  to 
be  quite  immune  to  experimental  inoculation,  though  it  can, 
under  certain  conditions,  contract  the  disease  by  natural  channels. 
The  guinea-pig  is  the  animal  most  convenient  for  experimental 
inoculation.  When  the  disease  is  set  up  in  the  guinea-pig  by 
subcutaneous  inoculation  with  garden  soil,  death  usually  occurs 
in  about  twenty-four  to  forty-eight  hours.  There  is  an  intense 
inflammatory  oedema  around  the  site  of  inoculation,  which 
extends  over  the  wall  of  the  abdomen  and  thorax.  The  skin 
and  subcutaneous  tissue  are  infiltrated  with  a  reddish  brown  fluid 
and  softened  ;  they  contain  bubbles  of  gas  and  are  at  places 
gangrenous.  The  superficial  muscles  are  also  involved.  These 
parts  have  a  very  putrid  odour.  The  internal  organs  are  con- 
gested, the  spleen  soft  but  not  much  enlarged.  In  such  con- 
ditions the  bacillus  of  malignant  oedema,  both  in  short  and  long 
forms,  will  be  found  in  the  affected  tissues  along  with  various 
other  organisms.  Spores  may  be  present,  especially  when  the 
examination  is  made  some  time  after  the  death  of  the  animal. 
If  the  animal  is  examined  immediately  after  death,  a  few  of  the 
bacilli  may  be  present  in  the  peritoneum  and  pleurae,  usually  in 
the  form  of  long  motile  filaments,  but  they  are  almost  invariably 
absent  from  the  blood.  A  short  time  after  death,  however,  they 
spread  directly  into  the  blood  and  various  organs,  and  may  then 
be  found  in  considerable  numbers. 

Subcutaneous  inoculation  with  pure  cultures  of  the  bacillus  of 
malignant  oedema  produces  chiefly  a  spreading  bloody  oedema, 
the  muscles  being  softened  and  partly  necrosed ;  but  there  is 
little  formation  of  gas,  and  the  putrid  odour  is  almost  absent. 


BACILLUS   BOTULINUS  393 

When  the  bacilli  are  injected  into  mice,  however,  they  enter 
and  multiply  in  the  blood  stream,  and  they  are  found  in  con- 
siderable numbers  in  the  various  organs,  so  that  a  condition  not 
unlike  that  of  anthrax  is  found.  The  spleen  also  is  much 
swollen. 

The  virulence  of  the  bacillus  of  malignant  oedema  varies  con- 
siderably in  different  cases,  and  it  always  becomes  diminished  in 
cultures  grown  for  some  time.  To  produce  a  fatal  disease,  a 
relatively  large  number  of  the  organisms  is  necessary,  and  these 
must  be  introduced  deeply  into  the  tissues,  inoculation  by  scari- 
fication being  followed  by  no  result.  A  smaller  dose  produces  a 
fatal  result  when  injected  along  with  various  other  organisms 
(bacillus  prodigiosus,  etc.). 

Immunity. — Malignant  oedema  was  one  of  the  first  diseases 
against  which  immunity  was  produced  by  injections  of  toxins. 
The  filtered  cultures  of  the  bacillus  in  sufficient  doses  produce 
death  with  the  same  symptoms  as  those  caused  by  the  living 
organisms,  but  a  relatively  large  quantity  is  necessary.  Chamber- 
land  and  Iloux  (1887)  found  that  if  guinea-pigs  were  injected 
with  several  non-fatal  doses  of  cultures  sterilised  by  heat  or  freed 
from  the  bacilli  by  filtration,  immunity  against  the  living  organism 
could  be  developed  in  a  comparatively  short  time.  They  found 
that  the  filtered  serum  of  animals  dead  of  the  disease  is  more 
highly  toxic,  and  also  gives  immunity  when  injected  in  small 
doses.  These  experiments  have  been  confirmed  by  Sanfelice. 

Methods  of  Diagnosis. — In  any  case  of  supposed  malignant 
oedema,  the  fluid  from  the  affected  tissues  ought  first  to  be 
examined  microscopically,  to  ascertain  the  characters  of  the 
organisms  present.  Though  it  is  not  possible  to  identify  ab- 
solutely the  bacillus  of  malignant  oedema  without  cultivating  it, 
the  presence  of  spore-bearing  bacilli  with  the  characters  described 
above  is  highly  suspicious  (Fig.  1 29).  In  such  a  case  the  fluid 
containing  the -bacilli  should  be  first  exposed  to  a  temperature 
of  80°  C.  for  half  an  hour,  and  then  a  deep  glucose  agar  tube 
should  be  inoculated.  In  this  way  the  spore-free  organisms  are 
killed  off.  Pure  cultures  may  be  thus  obtained,  or  this  procedure 
may  require  to  be  followed  by  the  roll -tube  method  under 
anaerobic  conditions.  An  inoculation  experiment,  if  available, 
may  also  be  made  on  a  guinea-pig. 

BACILLUS  BOTULINUS. 

The  term  "  meat-poisoning  "  embraces  a  number  of  conditions 
produced  by  different  agents,  and  the  relation  of  the  bacillus  of 


394  BACILLUS   BOTULINUS 

Gaertner  to  one  class  of  case  has  already  been  discussed.  Another 
group  was  shown  by  van  Ermengem  in  1896  to  be  caused  by  an 
anaerobic  bacillus  to  which  he  gave  the  name  bacillus  botulinus. 
He  cultivated  the  organism  from  a  sample  of  ham,  the  ingestion 
of  which  in  the  raw  condition  had  produced  a  number  >of  cases 
of  poisoning,  some  of  them  followed  by  fatal  result.  The 
symptoms  in  these  cases  closely  corresponded  with  those  occur- 
ring in  the  so-called  "  sausage  poisoning  "  met  with  from  time  to 
time  in  Germany  and  other  countries  where  sausages  and  ham 
are  eaten  in  an  imperfectly  cooked  condition.  Such  cases  form 
a  fairly  well-defined  group,  the  symptoms  in  which  are  chiefly 
referable  to  an  action  on  the  medulla,  and,  as  will  be  detailed 
below,  similar  symptoms  have  been  experimentally  produced 
by  means  of  the  bacillus  mentioned  or  its  toxins.  The  chief 
symptoms  of  this  variety  of  botulismus,  as  detailed  by  van 
Ermengem,  are  disordered  secretion  in  the  mouth  and  nose,  more 
or  less  marked  ophthalmoplegia,  externa  and  interna  (dilated 
pupil,  ptosis,  etc.),  dysphagia,  and  sometimes  aphagia  with 
aphonia,  marked  constipation  and  retention  of  urine,  and  in 
fatal  cases  interference  with  the  cardiac  and  respiratory  centres. 
Along  with  these  there  is  practically  no  fever  and  no  interference 
with  the  intellectual  faculties.  The  symptoms  commence  at 
earliest  twelve  to  twenty-four  hours  after  ingestion  of  the  poison. 
From  the  ham  in  question,  which  was  not  decomposed  in  the 
ordinary  sense,  van  Ermengem  obtained  numerous  colonies  of  this 
bacillus,  the  leading  characters  of  which  are  given  below.  It  may 
be  added  that  Romer  obtained  practically  the  same  results  as  van 
Ermengem  in  a  similar  condition,  and  that  the  bacillus  botulinus 
has  been  cultivated  by  Kempner  from  the  intestine  of  the  pig. 

Microscopical  and  Cultural  Characters. — The  organism  is  a 
bacillus  of  considerable  size,  measuring  4  to  9  />t  in  length  and 
*9  to  1*2  //,  in  thickness;  it  has  somewhat  rounded  ends  and 
sometimes  is  seen  in  a  spindle  form.  It  is  often  arranged  in 
pairs,  sometimes  in  short  threads.  Under  certain  conditions  it 
forms  spores  which  are  oval  in  shape,  usually  terminal  in  position, 
and  a  little  thicker  than  the  bacilli.  It  is  a  motile  organism 
and  has  4  to  8  lateral  flagella  of  wavy  form.  It  stains  readily 
with  the  ordinary  dyes,  and  also  retains  the  colour  in  Gram's 
method,  though  care  must  be  employed  in  decolorising. 

The  organism  can  be  readily  cultivated  on  the  ordinary 
media,  but  only  under  strictly  anaerobic  conditions.  In  glucose 
gelatin  a  whitish  line  of  growth  forms  with  lateral  offshoots, 
but  liquefaction  with  abundant  gas  formation  soon  occurs.  In 
gelatin  plates  the  colonies  after  four  to  six  days  are  somewhat 


MICROSCOPICAL  AND  CULTURAL  CHARACTERS  395 

characteristic ;  they  appear  to  the  naked  eye  as  small  semi- 
transparent  spheres,  and  these  on  examination  under  a  low 
power  of  the  microscope  have  a  yellowish-brown  colour  and  are 
seen  to  be  composed  of  granules  which  show  a  streaming  move- 
ment, especially  at  the  periphery.  Cultures  in  glucose  agar 
resemble  those  of  certain  other  anaerobes;  there  is  abundant 
development  of  gas,  and  the  medium  is  split  up  in  various 
directions.  The  cultures  have  a  rancid,  though  not  foul,  odour, 
due  chiefly  to  the  development  of  butyric  acid.  The  optimum 
temperature  is  below  that  of  the  body,  viz.,  between  20°  and 
30°  C. ;  at  the  body  temperature  growth  is  slower  and  less 
abundant  and  spore  formation  does  not  occur. 

Pathogenic  Effects.  —  Like  the  tetanus  bacillus  the  bacillus 
botulinus  has  little  power  of  flourishing  in  the  tissues,  whereas  it 
produces  a  very  powerful  toxin.  Van  Ermengem  found  that  the 
characteristic  symptoms  could  be  produced  in  certain  animals 
by  administering  watery  extracts  of  the  infected  ham  or  cultures 
either  by  the  alimentary  canal  or  by  subcutaneous  injection. 
Here  also  there  is  a  period  of  incubation  of  not  less  than  six  to 
twelve  hours  before  the  symptoms  appear,  and  when  the  dose  is 
small  a  somewhat  chronic  condition  may  result  in  which  local 
paralyses  form  a  striking  feature.  The  characteristic  effects 
can  also  be  produced  by  means  of  the  filtered  toxin  by  either  of 
the  methods  mentioned,  though  in  the  case  of  administration  by 
the  alimentary  canal  the  dose  requires  to  be  larger.  Here  also, 
as  in  the  case  of  the  tetanus  poison,  the  potency  of  the  toxin  is 
remarkable,  the  fatal  dose  for  a  guinea-pig  of  250  grm.  weight 
being  in  some  instances  '0005  c.c.  of  the  filtered  toxin.  In  cases 
of  poisoning  in  the  human  subject  the  effects  would  accordingly 
appear  to  be  produced  by  absorption  of  the  toxin  from  the 
alimentary  canal ;  it  is  only  after  or  immediately  before  death 
that  a  few  bacilli  may  enter  the  tissues.  Van  Ermengem 
obtained  a  few  colonies  from  the  spleen  of  a  patient  who  had 
died  from  ham-poisoning.  The  properties  of  the  botulinus  toxin 
have  been  investigated  and  have  been  found  to  correspond 
closely,  as  regards  relative  instability,  conditions  of  precipitation, 
etc.,  with  the  toxins  of  diphtheria  and  tetanus.  An  antitoxin 
has  also  been  prepared  by  Kempner  by  the  usual  methods,  and 
has  been  shown  not  only  to  have  the  neutralising  property  but 
to  have  considerable  therapeutical  value  when  administered 
some  hours  after  the  toxin.  The  direct  combining  affinity  of 
the  toxin  for  the  central  nervous  has  been  demonstrated  by 
Kempner  and  Schepilewsky  by  the  same  methods  as  Wassermann 
and  Takaki  employed  in  the  case  of  the  tetanus  toxin.  The 


396  QUARTER-EVIL 

condition  of  the  nerve  cells  in  experimental  poisoning  with  the 
botulinus  toxin  has  been  investigated  independently  by  Marinesco 
and  by  Kempner  and  Pollack,  and  these  observers  agree  as  to 
the  occurrence  of  marked  degenerative  changes,  especially  in  the 
motor  cells  in  the  spinal  cord  and  medulla.  Marinesco  also 
observed  hypertrophy  and  proliferation  of  the  neuroglia  cells 
around  them. 

These  observations,  therefore,  show  that  in  one  variety  of 
meat-poisoning  the  symptoms  are  produced  by  the  absorption  of 
the  toxins  of  the  bacillus  botulinus  from  the  alimentary  canal, 
and,  as  van  Ermengem  points  out,  it  is  of  special  importance  to 
note  that  the  meat  may  be  extensively  contaminated  with  this 
bacillus  and  may  contain  relatively  large  quantities  of  its  toxins 
without  the  ordinary  signs  of  decomposition  being  present. 
The  production  of  an  extracellular  toxin  by  this  organism,  with 
extremely  potent  action  on  the  nervous  system,  is  a  fact  of  great 
scientific  interest  and  has  a  bearing  on  the  etiology  of  other 
obscure  nervous  affections. 


QUARTER-EVIL  (GERMAN,  RAUSCHBRAND  ;  FRENCH,  CHARBON 
SYMPTOMATIQUE). 

The  characters  of  the  bacillus  need  be  only  briefly  described,  as,  so 
far  as  is  known,  it  never  infects  the  human  subject.  Tlie  natural  disease, 
which  specially  occurs  in  certain  localities,  affects  chiefly  sheep,  cattle, 
and  goats.  Infection  takes  place  by  some  wound  of  the  surface,  and 
there  spreads  in  the  region  around,  inflammatory  swelling  attended  by 
bloody  oedema  and  emphysema  of  the  tissues.  The  part  becomes  greatly 
swollen,  and  of  a  dark,  almost  black,  colour.  Hence  the  name  "  black- 
leg" by  which 'the  disease  is  sometimes  known.  The  bacillus  which 
produces  this  condition  is  present  in  large  numbers  in  the  affected  tissues, 
associated  with  other  organisms,  and  also  occurs  in  small  numbers  in 
the  blood  of  internal  organs. 

The  bacillus  morphologically  closely  resembles  that  of  malignant 
oedema.  Like  the  latter,  also,  it  is  a  strict  anaerobe,  and  its  conditions 
of  growth  as  regards  temperature  are  also  similar.  It  is,  however,  some- 
what thicker,  and  does  not  usually  form  such  long  filaments.  Moreover 
the  spores,  which  are  of  oval  shape  and  broader  than  the  bacillus,  are 
almost  invariably  situated  close  to  one  extremity,  though  not  actually 
terminal  (Fig.  132).  The  characters  of  the  cultures,  also,  resemble 
those  of  the  bacillus  of  malignant  oedema,  but  in  a  stab  culture  in 
glucose  agar  there  are  more  numerous  and  longer  lateral  offshoots,  the 
growth  being  also  more  luxuriant  (Fig.  131,  c).  This  bacillus  is  actively 
motile,  and  possesses  numerous  lateral  flagella. 

The  disease  can  be  readily  produced  in  various  animals,  e.g.  guinea- 
pigs,  by  inoculation  with  the  affected  tissues  of  diseased  animals,  and 
also  by  means  of  pure  cultures,  though  an  intramuscular  injection  of  a 
considerable  amount  of  the  latter  is  sometimes  necessary.  The  condition 
produced  in  this  way  closely  resembles  that  in  malignant  cedema,  though 


BACILLUS   ^ROGENES   CAPSULATUS 


397 


there  is  said  to  be  more  formation  of  gas  in  the  tissues.  Rabbits  are 
very  immune  against  this  disease,  whilst  they  are  comparatively  suscep- 
tible to  malignant  osdema.  As  in  the  case  of  tetanus,  inoculation  with 
living  spores  which  have  been  deprived  of  adherent  toxin  by  heat  does 
not  produce  the  disease.  A  toxin  can  be  separated  by  nitration  from 
bouillon  cultures.  It  is  fairly  resistant  to  heat,  withstanding  two  hours 

at  70-75°  C.  without  being 
destroyed,  and  it  is  also 
very  rapid  in  its  action,  being 
capable  in  appropriate  dose 
of  killing  a  horse  in  five 
minutes. 

The  disease  is  one  against 
which  immunity  can  be 
readily  produced  in  various 
ways,  and  methods  of  pre- 
ventive inoculation  have  been 
adopted  in  the  case  of  animals 
liable  to  suffer  from  it.  This 
subject  was  specially  worked 
out  by  Arloing,  Oornevin,  and 
Thomas,  and  later  by  others. 
Immunity  may  be  produced 
by  injection  with  a  non-fatal 
dose  of  the  virus  (i.e.  the 
oedematous  fluid  found  in  the 
tissues  of  aifected  animals  and 
Fio.  132.-Bacillus  of  quarter- evil,  showing  which  contains  the  bacilli) 

spores.     From  a  culture  m  glucose  agar,    Qr  b      injection   with  larger 

incubated  for  three  clays  at  37    C.  quantities   of  the    virus    at- 

Stamed  with  weak  carbol-rachsm.  >00.     tenuated  by  heat>  drying,  etc. 

It  can  be  produced  also  by 

cultures  attenuated  by  heat  and  by  the  products  of  the  bacilli  obtained 
by  nitration  of  cultures.  An  antitoxin  has  been  produced  against  the 
toxins  of  the  bacillus,  and  a  method  of  protection  in  which  the  action 
of  this  antitoxin  is  combined  with  that  of  the  virus  has  been  used. 
The  antitoxin  is  said  to  increase  the  chemotactic  properties  of  the 
leucocytes. 


.BACILLUS  NEOCENES  CAPSULATUS. 

This  bacillus,  though  sometimes  aiding  in  the  production  of  patho- 
logical changes,  is  chiefly  of  interest  on  account  of  the  extensive  gaseous 
development  which  it  gives  rise  to  in  the  tissues  post  mortem.  It  was 
described  by  Welch  and  Nuttall  in  1892  ;  it  is  now  recognised  as  being 
identical  with  an  organism  found  in  gaseous  phlegmon  by  E.  Fraenkel, 
and  called  by  him  the  bacillus  phlegmones  emphysematosce.  The  organism 
is  a  comparatively  large  one,  measuring  3  to  6  /u,  in  length  and  having  a 
thickness  about  the  same  as  that  of  the  anthrax  bacillus  ;  its  ends  are 
square  or  slightly  rounded  (Fig.  133).  It  often  occurs  in  pairs,  sometimes  in 
chains  ;  occasionally  filamentous  forms  are  met  with.  It  usually  shows 
a  well-marked  capsule,  hence  the  name  ;  it  is  non-motile  and  does  not 
form  spores.  It  stains  readily  with  the  basic  aniline  dyes  and  retains 
the  stain  in  Gram's  method.  It  grows  readily  on  the  ordinary  media, 


398 


BACILLUS   ^ROGENES   CAPSULATUS 


but  only  under  anaerobic  conditions  ;  the  optimum  temperature  is  that 
of  the  body,  growth  at  the  room  temperature  being  comparatively  slow. 
In  a  puncture  culture  in  agar  there  is  an  abundant  whitish  line  of  growth 
with  somewhat  indented  margin  ;  the  individual  colonies  are  white  and 
of  rounded  or  oval  form.  There  is  practically  no  liquefaction  of  gelatin, 
though  this  medium  becomes  somewhat  softened  around  the  growth. 
In  all  cases  there  is  a  tendency  to  abundant  evolution  of  gas  in  the 

cultures,  and  this  is  especi- 
ally marked  when  ferment- 
able sugars  are  present. 

The  organism  appears  to 
be  the  most  frequent  cause 
of  rapid  gaseous  develop- 
ment in  the  blood  and 
organs  post  mortem,  this 
depending  upon  an  invasion 
of  the  blood  immediately 
before  death.  In  such  cases, 
even  within  twenty  -  four 
hours  under  ordinary  con- 
ditions, large  bubbles  of 
gas  may  be  present  in  the 
veins,  and  the  organs  may 
be  beset  with  gas-contain- 
ing spheres  of  various  sizes  ; 
the  liver  is  usually  the 
organ  most  affected,  and  its 

appearance   has  been  com- 
FIG.     133.  —  Bacillus     aerogeues    capsulatus  ;  d   to  tbat   of  Gruyere 

film   preparation  from    bone- marrow  in  a    c}ieese       The   invasion   by 
case  where  gas-cavities  were  present  in  the    this  organism  is  met  with 

from  time  to  time  in  puer- 
peral cases,  and  also  in 

connection  with  ulcerative  or  gangrenous  conditions  of  the  intestine  ; 
the  bacillus  is  also  found  not  infrequently  in  'the  peritoneum  in 
cases  of  perforation.  Although  the  striking  changes  in  the  organs 
are  due  to  a  post-mortem  development  of  the  bacillus,  there  is  no 
doubt  that  its  entrance  into  the  blood  stream  often  hastens  death, 
and  may  in  some  instances  be  the  cause  of  it.  As  already  stated,  the 
organism  is  also  met  with  in  some  cases  of  spreading  oedema  with 
emphysema  as  a  leading  feature. 

When  tested  experimentally  the  bacillus  by  itself  is  found  to  have 
little  pathogenic  action.  Injection  of  pure  cultures  in  rabbits  and 
guinea-pigs  may  be  followed  by  little  result,  but  sometimes  in  the  latter 
animals  "gaseous  phlegmon"  is  produced,  without  suppuration  unless 
other  organisms  are  present.  If  a  small  quantity  of  culture  be  injected 
intravenously,  e.g.  in  a  rabbit,  and  then  the  animal  be  killed,  bubbles 
of  gas  are  rapidly  produced  in  the  blood  and  organs,  the  picture  corre- 
sponding with  that  in  the  human  cases. 


1  \:  NIVERSITY 

or 


CHAPTER   XVII. 

CHOLERA. 

Introductory. — It  is  no  exaggeration  of  the  facts  to  say  that 
previously  to  1883  practically  nothing  of  value  was  known  regard- 
ing the  nature  of  the  virus  of  cholera.  In  that  year  Koch  was 
sent  to  Egypt,  where  the  disease  had  broken  out,  in  charge  of  a 
Commission  for  the  purpose  of  investigating  its  nature.  In  the 
course  of  his  researches  he  discovered  the  organism  now  generally 
known  as  the  "comma  bacillus"  or  the  "cholera  spirillum." 
He  also  obtained  pure  cultures  of  the  organism  from  a  large 
number  of  cases  of  cholera,  and  described  their  characters.  The 
results  of  his  researches  were  given  at  the  first  Cholera  Conference 
at  Berlin  in  1884. 

Since  Koch's  discovery,  and  especially  during  the  epidemic  in 
Europe  in  1892-93,  spirilla  have  been  cultivated  from  cases  of 
cholera  in  a  great  many  different  localities,  and  though  this 
extensive  investigation  has  revealed  the  invariable  presence  in 
true  cholera  of  organisms  resembling  more  or  less  closely  Koch's 
spirillum,  certain  difficulties  have  arisen.  For  it  has  been  found 
that  the  cultures  obtained  from  different  places  have  shown  con- 
siderable variations  in  their  characters,  and,  further,  spirilla 
which  closely  resemble  Koch's  cholera  spirillum  have  been 
cultivated  from  sources  other  than  cases  of  true  cholera.  There 
has  therefore  been  much  controversy,  on  the  one  hand,  as  to  the 
signification  of  these  variations, — whether  they  are  to  be  regarded 
as  indicating  distinct  species  or  merely  varieties  of  the  same 
species, — and  on  the  other  hand,  as  to  the  means  of  distinguishing 
the  cholera  spirillum  from  other  species  which  resemble  it.  These 
questions  will  be  discussed  below. 

In  considering  the  bacteriology  of  cholera  it  is  to  be  borne  in 
mind  that  in  this  disease,  in  addition  to  the  evidence  of  great 
intestinal  irritation,  accompanied  by  profuse  watery  discharge,  and 


400  .    CHOLERA 

often  by  vomiting,  there  are  also  symptoms  of  general  systemic 
disturbance  which  cannot  be  accounted  for  merely  by  the  with- 
drawal of  water  and  certain  substances  from  the  system.  Such 
symptoms  include  the  profound  general  prostration,  cramps  in 
the  muscles,  extreme  cardiac  depression,  the  cold  and  clammy 
condition  of  the  surface,  the  subnormal  temperature,  suppression 
of  urine,  etc.  These  taken  in  their  entirety  are  indications  of  a 
general  poisoning  in  which  the  circulatory  and  thermo-regulatory 
mechanisms  are  specially  involved.  In  some,  though  rare,  cases 
known  as  cholera  sicca,  general  collapse  occurs  with  remarkable 
suddenness,  and  is  rapidly  followed  by  a  fatal  result,  whilst  there 
is  little  or  no  evacuation  from  the  bowel,  though  post  mortem  the 

intestine    is     distended 

^-Ai*    \  with  fluid  contents.     As 

*$*'&£  *"^    >  *ke  characteristic  organ- 

^   "  ^J  **j  *L+.  isms  in  cholera  are  found 

/ "          *c  ^Vt  ^  xf  *4L.>  '  1*""*     £          on^  *n  ^e  ifltestine,  the 

* *f  S^Q..C'O'C        general  disturbances  are 

f>  -v  j«3^H      to    be  regarded   as   the 
•-*.cf~(X_^  -V,^  •  •  1A.  _f°    •    __,    , 


r  ^  ;  ^      ^  ? ' 

."t  -  *  absorbed  from  the  bowel . 

r^          yf^T^-  It  is  also  to  be  noted 

v.'^'V     ^^  r  ~;  f  -a  r^r  5^  that  cholera  is  a  disease 

^"**'>     j  ^  ^f.  •""**  of  which  the  onset  and 

.    i5x*  l*vf|   ^        '  '  ^  -r  r-  course    are    much  more 

^'(--w^  $3  j*f~   <  rapid  than  is  the  case  in 

^J**,"*  most   infective  diseases, 

•*  *r      ,"  such    as     typhoid    and 

FIG.  134.-Cholera  spirilla,  from  a  culture  on    diphtheria  :      and      that 

agar  of  twenty-four  hours'  growth.  recovery    also,    when    it 

Stained  with  weak  carbol-fuchsin.      x  1000.      takes  place,  does  so  more 

quickly.  The  two  factors 

to  be  correlated  to  these  facts  are  (a)  a  rapid  multiplication  of 
organisms,  (b)  the  production  of  rapidly  acting  toxins. 

The  Cholera  Spirillum.  Microscopical  Characters. — The 
cholera  spirilla  as  found  in  the  intestines  in  cholera  are  small 
organisms  measuring  about  1*5  to  2  JJL  in  length,  and  rather  less 
than  *5  in  thickness.  They  are  distinctly  curved  in  one  direction, 
hence  the  appearance  of  a  comma  (Fig.  134)  ;  most  occur  singly, 
but  some  are  attached  in  pairs  and  curved  in  opposite  directions, 
so  that  an  S-shape  results.  Longer  forms  are  rarely  seen  in  the 
intestine,  but  in  cultures  in  fluids,  as  is  especially  well  seen  in 
hanging-drop  preparations,  they  may  grow  into  longer  spiral 
filaments,  showing  a  large  number  of  turns.  In  film  preparations 


THE   CHOLERA   SPIRILLUM 


401 


FIG.  135. — Cholera  spirilla  stained  to  show 
the  terminal  flagella.      x  1000. 


made  from  the  intestinal  contents  in  typical  cases,  these  organ- 
isms are  present  in  enor- 
mous numbers  in  almost 
pure  culture,  most  of  the 
spirilla  lying  with  their 
long  axes  in  the  same 
direction,  so  as  to  give  the 
appearance  which  Koch 
compared  to  a  number  of 
fish  in  a  stream. 

They  possess  very  active 
motility,  which  is  most 
marked  in  the  single  forms. 
When  stained  by  the  suit- 
able methods  they  are  seen 
to  be  flagellated.  Usually 
a  single  terminal  flagellum 
is  present  at  one  end 
only  (Fig.  135).  It  is 
very  delicate,  and  measures 

four  or  five  times  the  length  of  the  organism.  In  some  varieties, 
however,  there  may  be  a  flagellum  at  both  ends,  or  more  than 
one  may  be  present ;  cul- 
tures obtained  at  different 
places  have  shown  con- 
siderable variations  in  this 
respect.  Cholera  spirilla 
do  not  form  spores.  In 
old  cultures  the  organisms 
may  present  great  variety 
in  size  and  shape.  Some 
are  irregularly  twisted  fila- 
ments, sometimes  globose, 
sometimes  clubbed  at  their 
extremities,  and  also  show- 
ing irregular  swellings  along 
their  course.  Others  are 
short  and  thick,  and  may 
have  the  appearance  of  large  FIG.  136.— Cholera  spirilla  from  an  old  agar 
COCci,  Often  Staining  faintly.  culture>  sh°wi»g  irregularities  in  size  and 
Ari  .  •.  i  .  shape,  with  numerous  faintly -stained 

All  these  changes  m  appear-        coc£oid  bodies_invoiution  forms. 

ance  are  to  be  classed  to-  stained  with  fuchsin.     x  1000. 

gether  as  involution  forms. 

Staining. — Cholera  spirilla  stain  readily  with  the  usual  basic 
26 


402  CHOLERA 

aniline  stains,  though  Loffler's  methylene-blue  or  weak  carbol- 
fuchsin  is  specially  suitable.  They  lose  the  stain  in  Gram's 
method. 

Distribution  within  the  Body. — The  chief  fact  in  this  con- 
nection is  that  the  spirilla  are  confined  to  the  intestine,  and  are 
not  present  in  the  blood  or  internal  organs.  This  was  determined 
by  Koch  in  his  earlier  work,  and  his  statement  has  been  amply 
confirmed.  In  cases  in  which  there  is  the  characteristic  "  rice- 
water  "  fluid  in  the  intestines,  they  occur  in  enormous  numbers 
—almost  in  pure  culture.  The  lower  half  of  the  small  intestine 
is  the  part  most  affected.  Its  surface  epithelium  becomes  shed 
in  great  part,  and  the  flakes  floating  in  the  fluid  consist  chiefly 
of  masses  of  epithelial  cells  and  mucus,  amongst  which  are 
numerous  spirilla.  The  spirilla  also  penetrate  the  follicles  of 
Lieberkiihn,  and  may  be  seen  lying  between  the  basement 
membrane  and  the  epithelial  lining,  which  becomes  loosened  by 
their  action.  They  are,  however,  rarely  found  in  the  connective 
tissue  beneath,  and  never  penetrate  deeply.  Along  with  these 
changes  there  is  congestion  of  the  mucosa,  especially  around  the 
Peyer's  patches  and  solitary  glands,  which  are  somewhat  swollen 
and  prominent.  In  some  very  acute  cases  the  mucosa  may  show 
general  acute  congestion  with  a  rosy  pink  colour  but  very  little 
desquamation  of  epithelium,  the  intestinal  contents  being  a  com- 
paratively clear  fluid  containing  the  spirilla  in  large  numbers. 
In  other  cases  of  a  more  chronic  type,  the  intestine  may  show 
more  extensive  necrosis  of  the  mucosa  and  a  considerable 
amount  of  haemorrhage  into  its  substance,  along  with  formation 
of  false  membrane  at  places.  The  intestinal  contents  in  such 
cases  are  blood-stained  and  foul-smelling,  there  being  a  great 
proportion  of  other  organisms  present  besides  the  cholera  spirilla 
(Koch). 

Cultivation. — (For  Methods,  see  p.  413.) 
•  The  cholera  spirillum  grows  readily  on  all  the  ordinary  media, 
and  with  the  exception  of  that  on  potato,  growth  takes  place  at 
the  ordinary  room  temperature.  The  most  suitable  temperature, 
however,  is  that  of  the  body,  and  growth  usually  stops  about 
16°  C.,  though  in  some  cases  it  has  been  obtained  at  a  lower 
temperature. 

Peptone  Gelatin. — On  this  medium  the  organism  grows  well 
and  produces  liquefaction.  In  puncture  cultivations  at  22°  C.  a 
whitish  line  appears  along  the  needle  track,  at  the  upper  part  of 
which  liquefaction  commences,  and  as  evaporation  quickly  occurs, 
a  small  bell-shaped  depression  forms,  which  gives  the  appearance 
of  an  air-bubble.  On  the  fourth  or  fifth  day  we  get  the  following 


CULTIVATION 


403 


appearance :  there  is  at  the  surface  the  bubble -shaped  de- 
pression ;  below  this  there  is  a  funnel-shaped  area  of  liquefaction, 
the  fluid  being  only  slightly  turbid,  but  showing  at  its  lower  end 
thick  masses  of  growth  of  a  more  or  less  spiral  shape  (Fig.  137). 
The  liquefied  portion  gradually  tapers  off  downwards  towards  the 
needle  track.  (This  appearance  is,  however,  in  some  varieties  not 
produced  till  much  later,  especially  when 
the  gelatin  is  very  stiff,  and,  in  other 
varieties  which  liquefy  very  slowly,  may  not 
be  met  with  at  all.)  At  a  later  stage,  lique- 
faction spreads  and  may  reach  the  side  of 
the  tube. 

In  gelatin  plates  the  colonies  are  some- 
what characteristic.  They  appear  as  minute 
whitish  points,  visible  in  twenty-four  to  forty- 
eight  hours,  the  surface  of  which,  under  a  low 
power  of  the  microscope,  is  irregularly  granular 
or  furrowed  (Fig.  138,  A),  and  later  has  an 
appearance  which  has  been  compared  to 
fragments  of  broken  glass.  Liquefaction 
occurs,  and  the  colony  sinks  into  the  small 
cup  formed,  the  plate  then  showing  small 
sharply  -  marked  rings  around  the  colonies. 
Under  the  microscope  the  outer  margin  of 
the  cup  is  circular  and  sharply  marked. 
Within  the  cup  the  liquefied  portion  forms 
a  ring  which  has  a  more  or  less  granular 
appearance,  whilst  the  mass  of  growth  in  the 
centre  is  irregular  and  often  broken  up  at  its 
margins  (Fig.  138,  B).  The  growth  of  the 
colonies  in  gelatin  plates  constitutes  one  of 
the  most  important  means  of  distinguishing 
the  cholera  spirillum  from  other  organisms. 

On  the  surface  of  the  agar  media  a  thin 
almost  transparent  layer  forms,  which  pre- 
sents no  special  characters.  On  solidified 
blood  serum  the  growth  has  at  first  the  same 
appearance,  but  afterwards  liquefaction  of  the  medium  occurs. 
On  agar  plates  the  superficial  colonies  under  a  low  power  are 
circular  discs  of  brownish-yellow  colour,  and  more  transparent 
than  those  of  most  other  organisms.  On  potato  at  the  ordinary 
temperature,  growth  does  not  take  place,  but  on  incubation  at  a 
temperature  of  from  30°  to  37°  C.,  a  moist  layer  appears,  which 
assumes  a  dirty  brown  colour  somewhat  like  that  of  the  glanders 


FIG.  137.  —  Puncture 
culture  of  the  cholera 
spirillum  in  peptone 
gelatin  —  six  days' 
growth.  Natural  size. 


404  CHOLERA 

bacillus ;  the  appearance,  however,  varies  somewhat  in  different 
varieties,  and  also  on  different  sorts  of  potatoes. 

In  bouillon  with  alkaline  reaction  the  organism  grows  very 
readily,  there  occurring  in  twelve  hours  at  37°  C.  a  general 
turbidity,  while  the  surface  shows  a  thin  pellicle  composed  of 
spirilla  in  a  very  actively  motile  condition.  Growth  takes  place 
under  the  same  conditions  equally  rapidly  in  peptone  solution 
(1  per  cent  with  '5  per  cent  sodium  chloride  added). 

In  milk  also  the  organism  grows  well  and  produces  no  coagu- 
lation nor  any  change  in  its  appearance,  at  least  for  several  days. 

On  all  the  media  the  growth  of  the  cholera  spirillum  is  a 
relatively  rapid  one,  and  especially  is  this  the  case  in  peptone 


FIG.  138.  — Colonies  of  the  cholera  spirillum  in  a  gelatin  plate  ;  three  days' 
growth.  A  shows  the  granular  surface,  liquefaction  just  commencing  ;  in  B 
liquefaction  is  well  marked. 

solution  and  in  bouillon,  a  circumstance  of  importance  in  relation 
to  its  separation  in  cases  of  cholera  (vide  p.  413). 

The  cholera  organism  is  one  which  grows  much  more  rapidly 
in  the  presence  of  oxygen  than  in  anaerobic  conditions ;  in  the 
complete  exclusion  of  oxygen  very  little  growth  occurs. 

Cholera-red  reaction. — This  is  one  of  the  most  important  tests 
in  the  diagnosis  of  the  cholera  organism.  It  is  always  given  by 
a  true  cholera  spirillum,  and  though  the  reaction  is  not  peculiar 
to  it,  the  number  of  organisms  which  give  the  reaction  under 
the  conditions  mentioned  are  comparatively  few.  The  test  is 
made  by  adding  a  few  drops  of  pure  sulphuric  acid  to  a  culture 
in  bouillon  or  in  peptone  solution  (1  per  cent)  which  has  been 
incubated  for  twenty-four  hours  at  37°  C. ;  in  the  case  of  the 
cholera  spirillum  a  reddish-pink  colour  is  produced.  This  is  due 
to  the  fact  that  both  indol  and  a  nitrite  are  formed  by  the 
spirillum  in  the  medium.  The  addition  of  sulphuric  acid  causes 
a  nitroso-indol  body  to  be  produced  from  these,  and  this  gives 


POWERS   OF  RESISTANCE  405 

the  red  colour.  Here,  as  in  testing  for  the  production  of  indol 
by  other  bacteria,  it  is  found  that  not  every  specimen  of  peptone 
is  suitable,  and  it  is  advisable  to  select  a  peptone  which  gives 
the  characteristic  reaction  with  a  known  cholera  organism,  and 
to  use  it  for  further  tests.  It  is  also  essential  that  the  sulphuric 
acid  should  be  pure,  for  if  traces  of  nitrites  are  present  the 
reaction  might  be  given  by  an  organism  which  had  not  the 
power  of  forming  nitrites. 

Hcemolytic  Test. — This  method  introduced  by  Kraus  is 
performed  by  means  of  agar  plates,  a  small  quantity  of  sterile 
defibrinated  blood  being  added  to  the  agar  and  thoroughly 
diffused  ;  if  any  organism  has  hajmolytic  properties  a  clear  zone 
or  areola  forms  around  each  colony  by  the  diffusion  of  haemo- 
globin. In  no  instance  has  an  undoubted  cholera  organism  been 
found  to  produce  haemolysis,  whereas  many  species  of  spirilla 
closely  resembling  it  possess  marked  hsemolytic  action.  This 
test  may  accordingly  be  applied  along  with  the  others  in 
determining  the  identity  of  a  supposed  cholera  organism. 

Powers  of  Resistance.  —  In  their  resistance  against  heat, 
cholera  spirilla  correspond  with  most  spore-free  organisms,  and  are 
killed  in  an  hour  by  a  temperature  of  55°  C.,  and  much  more  rapidly 
at  higher  temperatures.  They  have  comparatively  high  powers 
of  resistance  against  great  cold,  and  have  been  found  alive  after 
being  exposed  for  several  hours  to  the  temperature  of  -  10°  C. 
They  are,  however,  killed  by  being  kept  in  ice  for  a  few  days. 
Against  the  ordinary  antiseptics  they  have  comparatively  low 
powers  of  resistance,  and  Pfuhl  found  that  the  addition  of  lime, 
in  the  proportion  of  1  per  cent,  to  water  containing  the  cholera 
organisms  was  sufficient  to  kill  them  in  the  course  of  an  hour. 

As  regards  the  powers  of  resistance  in  ordinary  conditions, 
the  following  facts  may  be  stated.  In  cholera  stools  kept  at  the 
ordinary  room  temperature,  the  cholera  organisms  are  rapidly 
outgrown  by .  putrefactive  bacteria,  but  in  exceptional  cases  they 
have  been  found  alive  even  after  two  or  three  months.  In  most 
experiments,  however,  attempts  to  cultivate  them  even  after  a 
much  shorter  time  have  failed.  The  general  conclusion  may  be 
drawn  from  the  work  of  various  observers  that  the  spirilla  do  not 
multiply  freely  in  ordinary  sewage  water,  although  they  may 
remain  alive  for  a  considerable  period  of  time.  On  moist  linen, 
as  Koch  showed,  they  can  flourish  very  rapidly.  When  the 
cholera  organisms  are  grown  along  with  other  organisms 
in  fluids  at  a  warm  temperature,  it  is  found  that  at  first  they 
may  multiply  more  rapidly  than  the  others,  but  that  after  a 
certain  time  they  are  outgrown  by  some  of  the  organisms  present, 


406  CHOLERA 

gradually  diminish  in  number,  and  ultimately  disappear.     It 
must  not,  however,  be  inferred  from  such  experiments  that  a 
similar  result  will  necessarily  follow  in  nature,  as  any  particular 
saprophytic  organism  requires  a  special  habitat — that  is,  certain 
suitable   conditions   for   its  growth  in  competition  with  other 
organisms.     Though  we  can  state  generally  that  the  conditions 
favourable  for  the  growth  of  the  cholera  spirillum  are  a  warm 
temperature,  moisture,  a  good  supply  of  oxygen,  and  a  consider- 
able proportion  of  organic  material,  we  do  not  know  the  exact 
circumstances  under  which  it  can  nourish  for  an  indefinite  period 
of  time  as  a  saprophyte.      The  fact  that  the  area   in   which 
cholera  is  an  endemic  disease  is  so  restricted  tends  to  show  that 
.the  conditions  for  a  prolonged  growth  of  the  spirillum  outside 
the  body  are  not  usually  supplied.     Yet,  on    the  other  hand, 
there  is  no    doubt  that  in  ordinary  conditions   it   can   live  a 
sufficient   time  outside  the  body   and  multiply  to  a  sufficient 
extent,  to  explain  all  the  facts  known  with  regard  to  the  per- 
sistence and  spread  of  cholera  epidemics. 

Numerous  experiments  show  that  the  cholera  organisms  are, 
as  a  rule,  rapidly  killed  by  drying,  usually  in  two  or  three 
minutes  when  the  drying  has  been  thorough,  and  it  is  inferred 
from  this  that  they  cannot  be  carried  in  the  living  condition  for 
any  great  distance  through  the  air,  a  conclusion  which  is  well 
supported  by  observations  on  the  spread  of  the  disease.  Cholera 
is  practically  always  transmitted  by  means  of  water  or  food 
contaminated  by  the  organism,  and  there  is  no  doubt  that  con- 
tamination of  the  water  supply  by  choleraic  discharges  is  the 
chief  means  by  which  areas  of  population  are  rapidly  infected. 
It  has  been  shown  that  if  flies  are  fed  on  material  containing 
cholera  organisms,  the  organisms  'may  be  found  alive  within  their 
bodies  twenty -four  hours  afterwards.  And  further,  Haffkine 
found  that  sterilised  milk  might  become  contaminated  with 
cholera  organisms,  if  kept  in  open  jars  to  which  flies  had  free 
access,  in  a  locality  infected  by  cholera.  It  is  quite  possible 
that  infection  may  be  carried  by  this  agency  in  some  cases. 

Experimental  Inoculation. — In  considering  the  effects  of 
inoculation  with  the  cholera  organism,  we  are  met  with  the 
difficulty  that  none  of  the  lower  animals,  so  far  as  is  known, 
suffer  from  the  disease  under  natural  conditions.  Even  in  places 
where  cholera  is  endemic,  no  corresponding  affection  has  been 
observed  in  any  animals.  And  further,  before  the  discovery  of 
the  cholera  organism,  various  efforts  had  been  made  to  induce 
the  disease  in  animals  by  feeding  them  with  cholera  dejecta,  but 
without  success.  It  is  therefore  not  surprising  that  the  earlier 


EXPERIMENTAL   INOCULATION  407 

« 

experiments  on  animals  by  feeding  them  with  pure  cultures  were 
attended  with  negative  results.  As  the  organisms  are  confined 
to  the  alimentary  tract  in  the  natural  disease,  attempts  to  induce 
their  multiplication  within  the  intestine  of  animals  by  artificially 
arranging  favouring  conditions,  have  occupied  a  prominent  place 
in  the  experimental  work.  We  shall  give  a  short  account  of 
such  experiments. 

Nikati  and  Bietsch  were  the  first  to  inject  the  organisms  directly  into 
the  duodenum  of  dogs  and  rabbits,  and  they  succeeded  in  producing,  in 
a  considerable  proportion  of  the  animals,  a  choleraic  condition  of  the 
intestine ;  in  their  earlier  experiments  the  common  bile  duct  was 
ligatured,  but  the  later  Avere  performed  without  this  operation.  These 
experiments  were  confirmed  by  other  observers,  including  Koch.  Think- 
ing that  probably  the  spirillum,  when  introduced  by  the  mouth,  is 
destroyed  by  the  action  of  the  hydrochloric  acid  of  the  gastric  secretion, 
Koch  first  neutralised  this  acidity  by  administering  to  guinea-pigs  5  c.c. 
of  a  5  per  cent  solution  of  carbonate  of  soda,  and  sometime  afterwards 
introduced  a  pure  culture  into  the  stomach  by  means  of  a  tube.  As  this 
method  failed  to  give  positive  results  he  tried  the  effect  of  artificially 
interfering  with  the  intestinal  peristalsis  by  injecting  tincture  of  opium 
into  the  peritoneum  (1  c.c.  per  200  grm.  weight),  in  addition  to  neutral- 
ising as  before  with  the  carbonate  of  sodium  solution.  The  result  was 
remarkable,  as  thirty  out  of  thirty -five  animals  treated  died.  The 
animals  infected  by  this  method  show  signs  of  general  prostration,  their 
posterior  extremities  being  especially  weakened  ;  the  abdomen  becomes 
tumid,  respiration  slow,  heart's  action  weak,  and  the  surface  cold. 
Death  occurs  after  a  few  hours.  Post  mortem  the  small  intestine  is 
distended,  its  mucous  membrane  congested,  and  it  contains  a  colourless 
fluid  with  small  flocculi  and  the  cholera  organisms  in  practically  pure 
cultures.  These  experiments,  which  have  been  repeatedly  confirmed, 
therefore  demonstrated  that  the  cholera 'organisms  could,  under  certain 
conditions,  set  up  in  animals  a  condition  in  some  respects  resembling 
cholera.  Koch,  however,  found  that  when  the  spirilla  of  Finkler  and 
Prior,  of  Deneke,  and  of  Miller  (vide  infra},  were  employed  by  the  same 
method,  a  certain,  though  much  smaller,  proportion  of  the  animals  died 
from  an  intestinal  infection.  Though  the  changes  in  these  cases  were 
not  so  characteristic,  they  were  sufficient  to  prevent  the  results  obtained 
with  the  cholera  organism  from  being  used  as  a  demonstration  of  the 
specific  relation  of  the  latter  to  the  disease. 

Within  later  years  some  additional  facts  of  high  interest  have  been 
established  with  regard  to  choleraic  infection  of  animals.  For  example, 
Sabolotny  found  that  in  the  marmot  an  intestinal  infection  readily  takes 
place  by  simple  feeding  with  the  organism,  there  resulting  the  usual 
intestinal  changes,  sometimes  with  hsemorrhagic  peritonitis,  the  organ- 
isms, however,  being  present  also  in  the  blood.  It  was  found  by  Issaeff 
and  Kolle  that  young  rabbits  could  be  infected  by  merely  neutralising 
the  gastric  secretion  with  sodium  carbonate,  the  use  of  opium  being 
unnecessary  ;  but  of  special  interest  is  the  fact,  discovered  by  Metclmikoff, 
that  in  the  case  of.  young  rabbits  shortly  after  birth  a  large  proportion 
die  of  choleraic  infection  when  the  organisms  are  simply  introduced 
along  with  the  milk,  as  may  be  done  by  infecting  the  teats  of  the 
mother.  Further,  from  these  animals  thus  infected  the  disease  may  be 


408  CHOLERA 

transmitted  to  others  by  a  natural  mode  of  infection.  In  this  affection 
of  young  rabbits  many  of  the  symptoms  of  cholera  are  present.  The 
organisms  occur  in  large  numbers  in  the  intestine,  and  in  some  cases  a 
few  may  be  found  in  the  blood,  and  especially  in  the  gall  bladder.  Many 
of  these  experiments  were  performed  with  the  vibrio  of  Massowah,  which 
is  now  admitted  not  to  be  a  true  cholera  organism,  others  with  a 
cholera  vibrio  obtained  from  the  water  of  the  Seine. 

It  will  be  seen  from  the  above  account  that  the  evidence 
obtained  from  experiments  on  intestinal  infection  of  animals, 
though  by  no  means  sufficient  to  establish  the  specific  relation- 
ship of  the  cholera  organism,  is  on  the  whole  favourable  to  this 
view,  especially  when  it  is  borne  in  mind  that  animals  do  not  in 
natural  conditions  suffer  from  the  disease. 

Experiments  performed  by  direct  inoculation  also  supply 
interesting  facts.  Intraperitoneal  injection  in  guinea-pigs  is 
followed  by  general  symptoms  of  illness,  the  most  prominent 
being  distension  of  the  abdomen,  subnormal  temperature,  and, 
ultimately,  profound  collapse.  There  is  peritoneal  effusion, 
which  may  be  comparatively  clear,  or  may  be  somewhat  turbid 
and  contain  flakes  of  lymph,  according  to  the  stage  at  which 
death  takes  place.  If  the  dose  is  large,"  organisms  are  found 
in  considerable  numbers  in  the  blood  and  also  in  the  small 
intestine,  but  with  smaller  doses  they  are  practically  confined  to 
the  peritoneum.  Kolle  found  that  when  the  minimum  lethal 
dose  was  used  in  guinea-pigs,  the  peritoneum  might  be  free  from 
organisms  at  the  time  of  death,  the  fatal  result  having  taken 
place  from  an  intoxication  (cf.  diphtheria,  p.  360).  These  and 
other  experiments  show  that  though  the  organisms  undergo  a 
certain  amount  of  multiplication  when  introduced  by  the 
channels  mentioned,  still  the  tendency  to  invade  the  tissues  is 
not  a  marked  one.  On  the  other  hand  the  symptoms  of  general 
intoxication  are  always  pronounced.  Hence  arise  questions  as 
to  the  nature  and  mode  of  action  of  toxic  bodies  produced  by 
the  cholera  organism. 

Toxins. — Though  there  is  no  doubt  that  there  are  formed  by 
Koch's  spirillum  toxic  bodies  which  produce  many  of  the 
symptoms  of  cholera,  there  is  at  present  very  little  satisfactory 
knowledge  regarding  their  chemical  nature.  The  following 
summary  may  be  given. 

It  has  been  shown,  especially  by  R.  Pfeiffer,1  that  toxic 
phenomena  can  be  produced  by  injection  of  the  dead  spirilla 
into  animals,  A  certain  quantity  of  a  young  culture  on  agar, 

1  Pfeiffer  obtained  his  earlier  results  with  a  vibrio  from  Massowah,  which  is 
now  known  (as  mentioned  above)  not  to  be  a  true  cholera  organism.  This  fact 
shows  that  the  effects  described  are  not  specific  to  the  latter. 


TOXINS   OF   KOCH'S   SPIRILLUM  409 

killed  by  exposure  to  the  vapour  of  chloroform,  when  injected 
intraperitoneally  into  a  guinea-pig,  may  cause  death  in  from 
eight  to  twelve  hours.  There  is  extreme  collapse,  sometimes 
clonic  spasms  occur,  and  the  temperature  may  fall  below  30°  C. 
before  death.  Pfeiffer  considers  that  the  toxic  substances  are 
contained  in  the  bodies  of  the  organisms — that  is,  they  are  in- 
tracellular, — and  that  they  are  only  set  free  by  the  disintegration 
of  the  latter.  This  opinion  is  grounded  chiefly  on  the  fact  that 
when  bouillon  cultures  were  filtered,  he  found  that  the  filtrate 
possessed  very  feeble  toxic  properties.  He  showed  also  that 
when  an  animal  is  inoculated  intraperitoneally  with  the  cholera 
organism,  and  then  some  time  later  anti-cholera  serum  which 
produces  bacteriolysis  is  injected,  rapid  collapse  with  a  fatal 
result  may  ensue,  apparently  due  to  the  liberation  of  the  intra- 
cellular  toxins.  The  dead  cultures  administered  by  the  mouth 
produce  no  effect  unless  the  intestinal  epithelium  is  injured,  in 
which  case  poisoning  may  result.  He  considers  that  the 
desquamation  of  the  epithelium  is  an  essential  factor  in  the 
production  of  the  phenomena  of  the  disease  in  the  human 
subject.  Pfeiffer  found  that  the  toxic  bodies  were  to  a  great 
extent  destroyed  at  60°  C.,  but  even  after  heating  at  100°  C.  a 
small  proportion  of  toxin  remained,  which  had  the  same  physio- 
logical action.  Recently  A.  Macfadyen  found  that  the  product 
obtained  by  grinding  up  the  spirilla  frozen  by  means  of  liquid  air 
had  a  very  high  degree  of  toxicity  when  injected  intravenously. 
Like  Pfeiffer  he  found  that  the  "  endotoxin  "  was  in  great  part 
destroyed  at  60°  C. 

On  the  other  hand,  other  observers  (Petri,  Ransom,  Klein, 
and  others)  have  obtained  toxic  bodies  from  filtered  cultures. 
Metclmikoff,  E.  Roux,  and  Taurelli-Salimbeni  have  demon- 
strated the  formation  of  such  diffusible  toxic  bodies  in  fluid 
media.  By  means  of  cultures  placed  in  collodion  sacs  in  the 
peritoneum  of  animals,  they  found  that  the  living  organisms 
produce  toxic  bodies  which  diffuse  through  the  wall  of  the  sac 
and  produce  toxic  symptoms.  By  greatly  increasing  the 
virulence  of  the  organism,  then  growing  it  in  bouillon  and 
filtering  the  cultures  on  the  third  and  fourth  day,  they  obtained 
a  fluid  which  was  highly  toxic  to  guinea-pigs  (the  fatal  dose 
usually  being  i  c.c.  per  100  grm.  weight).  If  the  dose  of  the 
toxin  is  very  large,  death  follows  in  an  hour  or  even  less.  The 
symptoms  closely  resemble  those  obtained  by  Pfeiffer,  the  rapid 
fall  of  temperature  being  a  striking  feature.  They  found  that 
the  toxicity  of  the  filtrate  was  not  altered  by  boiling;  appar- 
ently the  toxic  substance  is  different  from  Pfeiffer's  endotoxin. 


410  CHOLERA 

Attempts  to  investigate  the  chemical  nature  of  the  toxic  bodies 
have  not  led  to  definite  results. 

Experiments  on  the  Human  Subject. — Experiments  have  also 
been  performed  in  the  case  of  the  human  subject,  both  intention- 
ally and  accidentally.  In  the  course  of  Koch's  earlier  work,  one 
of  the  workers  in  his  laboratory  shortly  after  leaving  was  seized 
with  severe  choleraic  symptoms.  The  stools  were  found  to 
contain  cholera  spirilla  in  enormous  numbers.  Recovery,  how- 
ever, took  place.  In  this  case  there  was  no  other  possible 
source  of  infection  than  the  cultures  with  which  the  man  had 
been  working,  as  no  cholera  was  present  in  Germany  at  the  time. 
Within  recent  years  a  considerable  number  of  experiments  have 
been  performed  on  the  human  subject,  which  certainly  show  that 
in  some  cases  more  or  less  severe  choleraic  symptoms  may  follow 
ingestion  of  pure  cultures,  whilst  in  others  no  effects  may  result. 
The  former  was  the  case,  for  example,  with  Emmerich  and 
Pettenkofer,  who  made  experiments  on  themselves,  the  former 
especially  becoming  seriously  ill.  In  the  case  of  both,  diarrhoea 
was  well  marked,  and  numerous  cholera  spirilla  were  present  in 
the  stools,  though  toxic  symptoms  were  proportionately  little 
pronounced.  Metchnikoff  also,  by  experiments  on  himself  and 
others,  obtained  results  which  convinced  him  of  the  specific 
relation  of  the  cholera  spirillum  to  the  disease.  Lastly,  we  may 
mention  the  case  of  Dr.  Orgel  in  Hamburg,  who  contracted  the 
disease  in  the  course  of  experiments  with  the  cholera  and  other 
spirilla,  and  died  in  spite  of  treatment.  It  is  believed  that  in 
sucking  up  some  peritoneal  fluid  containing  cholera  spirilla,  a 
little  entered  his  mouth  and  thus  infection  was  produced.  This 
took  place  in  September  1894  at  a  time  when  there  was  no 
cholera  in  Germany.  On  the  other  hand,  in  many  cases  the 
experimental  ingestion  of  cholera  spirilla  by  the  human  subject 
has  given  negative  results.  Still,  as  the  result  of  observation  of 
what  takes  place  in  a  cholera  epidemic,  it  is  the  general  opinion 
of  authorities  that  only  a  certain  proportion  of  people  are 
susceptible  to  cholera,  and  the  facts  mentioned  above  are,  in  our 
opinion,  of  the  greatest  importance  in  establishing  the  relation 
of  the  organism  to  the  disease. 

Immunity. — As  this  subject  is  discussed  later,  only  a  few 
facts  will  be  here  stated,  chiefly  for  the  purpose  of  making  clear 
what  follows  with  regard  to  the  means  of  distinguishing  the 
cholera  spirillum  from  other  organisms.  The  guinea-pig  or  any 
other  animal  may  be  .easily  immunised  against  the  cholera 
organism  by  repeated  injections  (conveniently  made  into  the 
peritoneum)  of  non-fatal  doses  of  the  spirilla.  It  is  better  to 


IMMUNITY  411 

commence  the  process  with  non-fatal  doses  of  cultures  killed  by 
the  vapour  of  chloroform  or  by  heat,  the  doses  being  gradually 
increased,  and  afterwards  to  proceed  with  increasing  doses  of  the 
living  organism.  In  this  way  a  high  degree  of  immunity  against 
the  organism  is  developed,  and  further,  the  blood  serum  of  an 
animal  thus  immunised  (anti-cholera  serum)  has  markedly  pro- 
tective power  when  injected,  even  in  a  small  quantity,  into  a 
guinea-pig  along  with  five  or  ten  times  the  fatal  dose  of 
the  living  organism.  Under  these  circumstances  the  spirilla 
undergo  a  granular  transformation  and,  ultimately,  solution  ;  this 
phenomenon  is  generally  known  as  Pfeiffer's  reaction,  and  was 
applied  by  him  to  distinguish  the  cholera  spirillum  from  organisms 
resembling  it.  The  following  are  the  details  : — 

Pfeiffer's  Reaction. — A  loopful  (2  rngrm.)  of  recent  agar  culture  of  the 
organism  to  be  tested  is  added  to  1  c.c.  of  ordinary  bouillon  containing 
'001  c.c.  of  anti-cholera  serum.  The  mixture  is  then  injected  into  the 
peritoneal  cavity  of  a  young  guinea-pig  (about  200  grm.  in  weight),  and 
the  peritoneal  fluid  of  this  animal  (conveniently  obtained  by  means  of 
capillary  glass  tubes  inserted  into  the  peritoneum)  is  examined  micro- 
scopically after  a  few  minutes.  If  the  spirilla  injected  have  been  cholera 
spirilla,  it  will  be  found  that  they  become  motionless,  swell  up  into 
globules,  and  ultimately  break  down  and  disappear — positive  result.  If 
they  are  found  active  and  motile,  then  the  possibility  of  their  being 
true  cholera  spirilla  may  be  excluded — negative  result.  In  the  former 
case  (positive  result)  there  is,  however,  still  the  possibility  that  the, 
organism  is  devoid  of  pathogenic  properties  and  has  been  destroyed  by 
the  normal  peritoneal  fluid.  A  control  experiment  should  accordingly 
be  made  with  *001  c.c.  of  normal  serum  in  place  of  the  anti-cholera  serum. 
If  no  alteration  of  the  organism  occurs  with  its  use,  then  it  is  to  be 
concluded  that  a  true  reaction  has  been  given. 

The  serum  of  an  animal  immunised  by  the  above  method  has 
also  marked  agglutinative  action  against  the  cholera  spirillum, 
and  this  property  closely  corresponds  with  Pfeiffer's  reaction  as 
regards  specificity.  Such  a  serum  has,  however,  little  protective 
effect  against  the  toxic  action  of  the  dead  spirilla.  On  the  other 
hand,  Macfadyen  by  injecting  the  endotoxin  derived  from  the 
spirilla  by  grinding  obtained  a  serum  which  had  antitoxic  as  well 
as  agglutinative  and  bacteriolytic  properties  (vide  Immunity). 
Metchnikoff  and  others  have  also  obtained  antitoxic  sera  which 
act  on  the  extra-cellular  toxins  obtained  by  filtration. 

The  serum  of  cholera  convalescents  has  been  found  to  possess 
properties  similar  to  those  of  immunised  animals ;  that  is,  it 
affords  protection  against  the  cholera  spirillum  and  may  also 
give  Pfeiffer's  reaction.  These  properties  of  the  serum  may  be 
present  eight  or  ten  days  after  the  attack  of  the  disease,  but 
are  most  marked  four  weeks  after ;  they  then  gradually  become 


412  CHOLERA 

weaker  and  disappear  in  two  or  three  months  (Pfeiffer  and 
Issaeff). 

Specific  agglutinative  properties  have,  however,  been  detected 
in  the  serum  of  cholera  patients  at  a  much  earlier  date,  in  some 
cases  even  on  the  first  day  of  the  disease,  though  usually  a  day 
or  two  later.  The  dilutions  used  were  1  : 15  to  1  : 120,  and  these 
had  no  appreciable  effect  on  organisms  other  than  the  cholera 
spirillum  (Achard  and  Bensande).  Nee'dless  to  say,  such  facts 
supply  strong  additional  evidence  of  the  relation  of  Koch's 
spirillum  to  cholera. 

Anti-Cholera  Inoculation. — Haffkine's  method  for  inoculation 
against  cholera  exemplifies  the  above  principles.  It  depends 
upon  (a)  attenuation  of  the  virus — that  is,  the  cholera  organism, 
and  (b)  exaltation  of  the  virus.  The  virulence  of  the  organism 
is  diminished  by  passing  a  current  of  sterile  air  over  the  surface 
of  the  cultures,  or  by  various  other  methods.  The  virulence  is 
exalted  by  the  method  of  passage — that  is,  by  growing  the 
organism  in  the  peritoneum  in  a  series  of  guinea-pigs/  By  the 
latter  method  the  virulence  after  a  time  is  increased  twenty-fold 
— that  is,  the  fatal  dose  has  been  reduced  to  a  twentieth  of  the 
original.  Cultures  treated  in  this  way  constitute  the  virus  ejraltr. 
Subcutaneous  injection  of  the  virus  exalte  produces  a  local 
necrosis,  and  may  be  followed  by  the  death  of  the  animal,  but  if 
the  animal  be  treated  first  with  the  attenuated  virus,  the  sub- 
sequent injection  of  the  virus  exalte  produces  only  a  local  oedema. 
After  inoculation  first  by  attenuated  and  afterwards  by  exalted 
virus,  the  guinea-pig  has  acquired  a  high  degree  of  immunity,  and 
Haffkine  believed  that  this  immunity  was  effective  in  the  case 
of  every  method  of  inoculation — that  is,  by  the  mouth  as  well  as 
by  injection  into  the  tissues.  After  trying  his  method  on  the 
human  subject  and  finding  it  free  from  risk,  he  extended  it  in 
practice  on  a  large  scale  in  India  in  1894,  and  these  experiments 
are  still  going  on.  So  far  the  results  are,  on  the  whole,  distinctly 
encouraging.  In  the  human  subject  two  or  sometimes  three  in- 
oculations were  formerly  made  with  attenuated  virus  before  the 
virus  exalte  was  used ;  now,  however,  a  single  injection  of  the 
latter  is  usually  practised.  Wassermann  and  Pfeiffer,  and  also 
Klein,  have  found  that  guinea-pigs  immunised  by  Haffkine's 
method  are  not  immunised  against  intestinal  infection  when  the 
animal  is  treated  by  Koch's  method  (vide  p.  407).  Notwith- 
standing this  fact  Haffkine's  method  may  still  have  a  beneficial 
effect,  though  it  may  not  be  preventive  in  all  cases. 

Methods  of  Diagnosis. — In  the  first  place,  the  stools  ought 
to  be  examined  microscopically.  Dried  film  preparations  should 


METHODS   OF   DIAGNOSIS  413 

be  made  and  stained  by  any  ordinary  stains,  though  carbol-fuchsin 
diluted  four  times  with  water  is  specially  to  be  recommended. 
Hanging-drop  preparations,  with  or  without  the  addition  of  a 
weak  watery  solution  of  gentian- violet  or  other  stain,  should  also 
be  made,  by  which  method  the  motility  of  the  organism  can  be 
readily  seen.  By  microscopic  examination  the  presence  of  spirilla 
will  be  ascertained,  and  an  idea  as  to  their  number  obtained. 
In  some  cases  the  cholera  spirilla  are  so  numerous  in  the  stools 
that  a  picture  is  presented  which  is  obtained  in  no  other  con- 
dition, and  a  microscopic  examination  may  be  sufficient  for 
practical  purposes.  According  to  Koch,  a  diagnosis  wTas  made 
in  50  per  cent  of  the  cases  during  the  Hamburg  epidemic  by 
microscopic  examination  alone.  In  the  case  of  the  first  appear- 
ance of  a  cholera-like  disease,  however,  all  the  other  tests 
should  be  applied  before  a  definite  diagnosis  of  cholera  is  made. 
Dunbar  has  recently  introduced  a  method  for  rapid  diagnosis 
which  depends  on  the  properties  of  an  anti-cholera  serum.  Two 
hang-drop  preparations  are  made,  each  consisting  of  a  small 
portion  of  mucus  from  the  suspected  stool  broken  up  in  peptone 
solution.  To  one  a  drop  of  a  50-fold  dilution  of  normal  serum 
is  added,  to  the  other  a  drop  of  a  500-fold  dilution  of  an  active 
cholera  serum.  If  the  spirilla  present  are  cholera  organisms 
they  retain  their  motility  in  the  first  preparation,  while  they  lose 
it  and  then  become  agglutinated  in  the  second.  By  this  method 
a  diagnosis  may  sometimes  be  given  in  a  few  minutes. 

If  the  organisms  are  very  numerous,  gelatin  or  agar  plates 
may  be  made  at  once  and  pure  cultures  obtained. 

If  the  spirilla  occur  in  comparatively  small  numbers,  the  best 
method  is  to  inoculate  peptone  solution  (1  per  cent)  and  incubate 
for  from  eight  to  twelve  hours.  At  the  end  of  that  time  the  spirilla 
will  be  found  on  microscopic  examination  in  enormous  numbers 
at  the  surface,  and  thereafter  plate  cultures  can  readily  be  made. 
If  the  spirilla  are  very  few  in  number,  or  if  a  suspected  water  is 
to  be  examined  for  cholera  organisms,  the  peptone  solution 
which  has  been  inoculated  should  be  examined  at  short  intervals 
till  the  spirilla  are  found  microscopically.  A  second  flask  of 
peptone  solution  should  then  be  inoculated,  and  possibly  again 
a  third  from  the  second.  By  this  method,  properly  carried  out, 
a  culture  may  be  obtained  which,  though  impure,  contains  a 
large  proportion  of  the  spirilla,  and  then  plate  cultures  may  be 
made. 

When  a  spirillum  has  been  obtained  in  pure  condition  by 
these  methods,  the  appearance  of  the  colonies  in  plates  should 
be  specially  noted,  the  test  for  the  cholera-red  reaction  should 


414  CHOLEEA 

be  applied,  and  in  many  cases  it  is  advisable  to  test  the  effects 
of  intraperitoneal  injection  of  a  portion  of  a  recent  agar  culture 
in  a  guinea-pig,  the  amount  sufficient  to  cause  death  being  also 
ascertained.  The  agglutinating  or  sedimenting  properties  of  the 
serum  of  the  patient  should  be  tested  against  a  known  cholera 
organism,  and  against  the  spirillum  cultivated  from  the  case. 
The  action  of  an  anti-cholera  serum,  i.e.  the  serum  of  animal 
immunised  against  the  cholera  spirillum,  should  be  tested  in  a 
similar  manner. 

Up  till  recent  times  there  had  been  cultivated  from  sources 
other  than  cholera  cases,  no  organism  which  gave  all  the  cultural 
and  biological  tests  (agglutination  and  Pfeiffer's  reaction)  of  the 
cholera  spirillum.  In  1905,  however,  Gotschlich  obtained  six 
different  strains  of  a  spirillum  which  conformed  in  all  these 
respects.  The  organisms  were  obtained  at  El  Tor  from  the 
intestines  of  pilgrims  who  had  died  with  dysenteric  symptoms, 
and  there  were  no  cases  of  cholera  in  the  vicinity.  The  organisms 
in  question,  however,  differ  from  the  cholera  organism  in  having 
marked  hsemolytic  action,  and  also  in  producing  a  rapidly  acting 
extra -cellular  toxin.  There  has  been  diversity  of  opinion 
with  regard  to  the  nature  of  these  organisms,  for  while  some 
consider  that  they  are  a  different  species  from  the  cholera 
organism,  others  regard  them  as  true  cholera  spirilla  which  had 
been  carried  by  the  patients,  although  no  symptoms  of  cholera 
resulted  from  their  presence.  If  they  are  not  to  be  regarded  as 
cholera  organisms,  we  have  the  striking  fact  that  they  correspond 
in  the  immunity  reactions.  This  instance  exemplifies  well  the 
great  difficulty  which  may  surround  the  identification  of  a 
particular  organism  obtained  from  non- cholera  cases,  and  one 
can  hardly  doubt  that  if  cholera -like  symptoms  had  been 
present  in  the  El  Tor  cases,  the  spirilla  would  have  been  accepted 
as  varieties  of  the  cholera  organism,  though  differing  in  their 
hsemolytic  action.  None  of  the  facts  ascertained,  however,  really 
affect  the  question  as  to  the  causal  relationship  of  Koch's  spirillum 
to  cholera,  although  they  indicate  the  difficulties  which  may 
attend  the  bacteriological  diagnosis  in  isolated  cases  of  the 
disease. 

General  Summary. — We  may  briefly  summarise  as  follows 
the  facts  in  favour  of  Koch's  spirillum  being  the  cause  of  cholera. 
First,  there  is  the  constant  presence  of  spirilla  in  true  cases  of 
cholera,  which  on  the  whole  conform  closely  with  Koch's 
description,  though  variations  undoubtedly  occur.  Moreover, 
the  facts  known  with  regard  to  their  conditions  of  growth,  etc., 
are  in  conformity  with  the  origin  and  spread  of  cholera  epidemics. 


SPIRILLA  RESEMBLING  CHOLERA  SPIRILLUM    415 

Secondly,  the  experiments  on  animals  with  Koch's  spirillum  or 
its  toxins  give  as  definite  results  as  one  can  reasonably  look  for 
in  view  of  the  fact  that  animals  do  not  suffer  naturally  from  the 
disease.  Thirdly,  the  experiments  on  the  human  subject  and 
the  results  of  accidental  infection  by  means  of  pure  cultures  are 
also  strongly  in  favour  of  this  view.  Fourthly,  the  agglutinative 
and  protective  properties  of  the  serum  of  cholera  patients  and 
convalescents  constitute  another  point  in  its  favour.  Fifthly, 
bacteriological  methods,  which  proceed  on  the  assumption  that 
Koch's  spirillum  is  the  cause  of  the  disease,  have  been  of  the 
greatest  value  in  the  diagnosis  of  the  disease.  And  lastly,  the 
results  of  Haffkine's  method  of  preventive  .inoculation  in  the 
human  subject,  which  are  on  the  whole  favourable,  also  supply 
additional  evidence.  If  all  these  facts  are  taken  together,  we 
consider  the  conclusion  must  be  arrived  at  that  the  growth  of 
Koch's  spirillum  in  the  intestine  is  the  immediate  cause  of  the 
disease.  This  does  not  exclude  the  probability  of  an  important 
part  being  played  by  conditions  of  weather  and  locality,  though 
such  are  very  imperfectly  understood.  Pettenkofer,  for  example, 
recognised  two  main  factors  in  the  causation  of  epidemics,  which 
he  designated  x  and  y,  and  considered  that  these  two  must  be 
present  together  in  order  that  cholera  may  spread.  The  x  is  the 
direct  cause  of  the  disease — an  organism  which  he  admitted 
to  be  Koch's  spirillum ;  the  y  includes  climatic  and  local  con- 
ditions, e.g.  state  of  ground-water,  etc. 

Other  Spirilla  resembling  the  Cholera  Organism. — These 
have  been  chiefly  obtained  either  from'  water  contaminated  by 
sewage  or  from  the  intestinal  discharge  in  cases  with  choleraic 
symptoms.  Some  of  them  differ  so  widely  in  their  cultural  and 
other  characters  (some,  for  example,  are  phosphorescent)  that  no 
one  would  hesitate  to  classify  them  as  distinct  species.  Others, 
however,  closely  resemble  the  cholera  organism. 

The  vibrio  berolinensis,  cultivated  by  Neisser  from  Berlin  sewage 
water,  differs  from  the  cholera  organism  only  in  the  appearance  of  its 
colonies  in  gelatin  plates,  its  weak  pathogenic  action,  and  its  giving  a 
negative  result  with  Pfeiffer's  test.  It,  however,  gives  the  cholera-red 
reaction.  The  vibrio  Danubicus,  cultivated  by  Heider  from  canal  water, 
also  differs  in  the  appearance  of  its  colonies  in  plates,  and  also  reacts 
negatively  to  Pfeiffer's  test  ;  in  most  respects  it  closely  resembles  the 
cholera  organism.  Another  spirillum  (v.  Ivanoff}  was  cultivated  by 
Ivanoff  from  the  stools  of  a  typhoid  patient  after  these  had  been  diluted 
with  water.  The  organism  differed  somewhat  in  the  appearance  of  its 
colonies  and  in  its  great  tendency  to  grow  out  in  the  form  of  long 
threads,  but  Pfeiffer  found  that  it  reacted  to  his  test  in  the  same  way  as 
the  cholera  organism,  and  he  considered  that  it  was  really  a  variety  of 
the  cholera  organism.  No  spirilla  could  be  found  microscopically  in  the 


416  CHOLERA 

stools  in  this  case,  and  Pfeiffer  is  of  the  opinion  that  the  organism 
gained  entrance  accidentally.  These  examples  will  show  how  differences 
of  opinion,  even  amongst  experts,  might  arise  as  to  whether  a  certain 
spirillum  were  really  the  cholera  organism  or  a  distinct  species  resem- 
bling it. 

A  few  examples  may  also  be  given  of  organisms  cultivated 
from  cases  in  which  cholera-like  symptoms  were  present. 

The  vibrio  of  Massowah  was  cultivated  by  Pasquale  from  a  case  during 
a  small  epidemic  of  cholera.  The  organism  so  closely  resembles  Koch's 
spirillum  that  it  was  accepted  by  several  authorities  as  the  true  cholera 
organism,  and,  as  already  stated,  Metchnikoff  produced  by  it  cholera 
symptoms  in  the  human  subject,  and  also  the  cholera-like  disease  in 
young  rabbits.  It  possesses  four  flagella,  has  a  high  degree  of  virulence, 
producing  septicaemia  both  in  guinea-pigs  and  pigeons,  and  its  colonies 
in  plates  differ  somewhat  from  the  cholera  organism.  Moreover,  it 
reacts  negatively  to  Pfeiffer's  test.  Another  organism,  the  v.  Gindha, 
wras  cultivated  by  Pasquale  from  a  well,  and  was  at  first  accepted  by 
Pfeiffer  as  the  cholera  organism,  but  afterwards  rejected,  chiefly  because 
it  failed  to  give  the  specific  immunity  reaction.  It  also  differs  some- 
what from  the  cholera  organism  in  its  pathogenic  effects,  and  it  fails  to 
give  the  cholera-red  reaction  or  gives  it  very  faintly. 

Pestana  and  Bettencourt  also  cultivated  a  species  of  spirillum  from  a 
number  of  cases  during  an  epidemic  in  Lisbon — an  epidemic  in  which 
there  were  symptoms  of  gas'tro- enteritis,  although  only  in  a  few  instances 
did  the  disease  resemble  cholera.  They  also  cultivated  the  same 
organism  from  the  drinking  water.  It  differs  from  the  cholera  organism 
in  the  appearance  of  its  colonies  and  of  puncture  cultures  in  gelatin.  It 
has  very  feeble  pathogenic  effects,  and  gives  a  very  faint,  or  no,  cholera- 
red  reaction.  To  Pfeiffer's  test  it  also  reacts  negatively.  Another 
spirillum  (v.  Romanus)  was  obtained  by  Celli  and  Santori  from  twelve 
out  of  forty-four  cases  where  there  were  the  symptoms  of  mild  cholera. 
This  organism  does  not  give  the  cholera-red  reaction,  nor  is  it  pathogenic 
for  animals.  They  look  upon  it  as  a  "  transitory  variety  "  of  the  cholera 
organism,  though  sufficient  evidence  for  this  view  is  not  adduced. 

We  have  mentioned  these  examples  in  order  to  show  some  of 
the  difficulties  which  exist  in  connection  with  this  subject.  It  is 
important  to  note  that,  on  the  one  hand,  spirilla  which  have 
been  judged  to  be  of  different  species  from  the  cholera  organism, 
have  been  cultivated  from  cases  in  which  cholera -like  symptoms 
were  present,  and,  on  the  other  hand,  in  cases  of  apparently  true 
cholera  considerable  variations  in  the  characters  of  the  cholera 
organisms  have  been  found.  Such  variations  have  especially 
been  recorded  by  Colonel  Cunningham  in  India.  It  is  there- 
fore quite  an  open  question  whether  some  of  the  organisms 
in  the  former  class  may  not  be  cholera  spirilla  which  have  under- 
gone variations  as  a  result  of  the  conditions  of  their  growth. 
That  such  variations  may  occur  we  have  a  considerable  amount 
of  evidence.  The  great  bulk  of  evidence,  however,  goes  to  show 


METCHNIKOFF'S   SPIRILLUM  41 Y 

that  Asiatic  cholera  always  spreads  as  an  epidemic  from  places 
in  India  where  the  disease  is  endemic,  and  that  its  direct  cause 
is  Koch's  spirillum.  It  is  sufficient  to  bear  in  mind  that  choleraic 
symptoms  may  be  produced  by  other  causes,  and  that  in  some 
of  such  cases  spirilla  which  have  some  resemblance  to  Koch's 
organism  may  be  present  in  the  intestinal  discharges,  though 
rarely  in  large  numbers. 

A  number  of  other  spirilla  have  been  cultivated,  which  are  of 
interest  on  account  of  their  points  of  resemblance  to  the  cholera 
organism,  though  probably  they  produce  no  pathological  condi- 
tions in  the  human  subject. 

Metchnikoff's  Spirillum  (vibrio  Metchnikovi). — This  organism  was  ob- 
tained by  Gamaleia  from  an  epidemic  disease  of  fowls  in  Odessa,  and  is  of 
special  interest  on  account  of  its  close  resemblance  to  the  cholera  organism. 
In  the  natural  disease,  which 
especially  affects  young 
fowls,  the  animals  suffer 
from  diarrhoea,  pass  into  a 

sort  of  stupor,  sitting  with  *^tf3   **•**  - 

their  feathers  ruffled,  and 
usually  die  within  forty-  ^ 
eight  hours.  The  intestines 
contain  a  greyish  -  yellow 
fluid,  sometimes  slightly 
blood-stained,  in  which  the 
spirilla  are  found.  A  few 
of  these  spirilla  may  also  be 
found  in  the  blood  in  the  v 5i*?~  *$"  ^&.  »  *%^  "^  A 

younger  fowls,  though 
generally  absent  from  the 
blood  in  the  older.  **',  "*(+{  *  m"  -  "*" 

Morphologically  the  or- 
ganism is  practically  identi- 
cal with  Koch's  spirillum 

(Fig.   139).      It  is  actively    FIG.    139. — Metchnikoff's    spirillum,  both    in 
motile,  and   has   the  same          curved  and  straight  forms  ;   from  an  agar 
staining      reactions.        Its          culture  of  twenty-four  hours' growth, 
growth  in  peptone  gelatin      Stained  with  weak  carbol-fuchsin.      x  1000. 
also  closely  resembles  that 

of  the  cholera  organism,  though  it  produces  liquefaction  more  rapidly 
(Fig.  140,  A).  In  gelatin  plates  the  young  colonies  are,  however, 
smoother  and  more  circular.  After  liquefaction  occurs,  some  of  the 
colonies  are  almost  identical  in  appearance  with  those  of  the  cholera 
vibrio,  whilst  others  show  more  uniformly  turbid  contents.  In  puncture 
cultures  the  growth  takes  place  more  rapidly,  but  in  appearance 
closely  resembles  that  of  the  cholera  organism  a  few  days  older.  Its 
growth  in  peptone  solution  too  is  closely  similar,  and  it  also  gives  the 
cholera-red  reaction. 

This  organism  can,  however,  be  readily  distinguished  from  the 
cholera  organism  by  the  effects  of  inoculation  on  animals,  especially  on 
pigeons  and  guinea-pigs.  Subcutaneous  inoculation  of  small  quantities 

27 


418 


CHOLERA 


of  pure  culture  in  pigeoiis  is  followed  by  septicaemia,  which  produces  a 
fatal  result  usually  within  twenty-four  hours.  Inoculation  with  the  same 
quantity  of  cholera  culture  produces  practically  no  result ;  even  with 
large  quantities  death  is  rarely  produced.  The  vibrio  Metehnikovi 
produces  somewhat  similar  effects  in  the  guinea-pig  to  those  in  the 

pigeon,  subcutaneous  inoculation 
being  followed  by  extensive  haemor- 
rhagic  osdema  and  a  rapidly  fatal 
septicaemia.  Young  fowls  can  be 
infected  by  feeding  with  virulent 
cultures.  We  have  evidence  from 
the  work  of  Gamaleia  that  the  toxins 
of  this  organism  have  somewhat  the 
same  action  as  those  of  the  cholera 
organism. 

The  organism  is  therefore  one 
which  very  closely  resembles  the 
cholera  organism,  the  results  on  in- 
oculating the  pigeon  offering  the  most 
ready  means  of  distinction.  It  gives 
a  negative  reaction  to  Pfeiffer's  test 
— that  is,  the  properties  of  an  anti- 
cholera  serum  are  not  exerted  against 
it.  It  may  also  be  mentioned  that 
an  organism  which  is  apparently  the 
same  as  the  vibrio  Metchnikovi  was 
cultivated  by  Pfuhl  from  water,  and 
named  v.  Nordhafen. 

Finkler  and  Prior's  Spirillum. — 
These  observers,  shortly  after  Koch's 
discovery  of  the  cholera  organism, 
separated  a  spirillum,  in  a  case  of 
cholera  nostras,  from  the  stools  after 
they  had  been  allowed  to  decompose 
for  several  days.  There  is,  however, 
no  evidence  that  the  spirillum  has 
any  causal  relationship  to  this  or  any 
other  disease  in  the  human  subject. 
Morphologically  it  closely  resembles 
Koch's  spirillum,  and  cannot  be  dis- 
tinguished from  it  by  its  micro- 
scopical characters,  although,  on  the 
whole,  it  tends  to  be  rather  thicker 
in  the  centre  and  more  pointed  at  the  ends  (Fig.  141).  In  cultures, 
however,  it  presents  marked  differences.  In  puncture  cultures  on 
gelatin  it  grows  much  more  quickly,  and  liquefaction  is  generally 
visible  within  twenty-four  hours.  The  liquefaction  spreads  rapidly, 
and  usually  in  forty  -  eight  hours  it  has  produced  a  funnel  -  shaped 
tube  with  turbid  contents,  denser  below  (Fig.  140,  B).  In  plate 
cultures  the  growth  of  the  colonies  is  proportionately  rapid.  Before 
they  have  produced  liquefaction  around  them,  they  appear,  unlike  those 
of  the  cholera  organism,  as  minute  spheres  with  smooth  margins.  When 
liquefaction  occurs,  they  appear  as  little  spheres  with  turbid  contents, 
which  rapidly  increase  in  size  ;  ultimately  general  liquefaction  occurs. 
On  potatoes  this  organism  grows  well  at  the  ordinary  temperature,  and 


FIG.  140. — Puncture  cultures  in 
peptone-gelatin. 

A.  MetchnikoflTs  spirillum.     Five 

days'  growth. 

B.  Finkler  and  Prior's  spirillum. 
Four  days'  growth.     Natural  size. 


DENEKE'S   SPIRILLUM  419 

in  two  or  three  days  has  formed  a  slimy  layer  of  greyish  -yellow  colour, 

which  rapidly  spreads  over  the  potato.     On  all  the  media  the  growth 

has  a  distinctly  foetid  odour.     A  growth  in  peptone  solution  fails  to  give 

the  cholera-red  reaction  at  the  end  of  twenty-four  hours,  though  later  a 

faint     reaction     may     ap  - 

pear.   As  stated  above,  Koch 

succeeded     in      producing, 

by  this  organism,  an  intes- 

tinal  affection  in  guinea-pigs  _ 

after  neutralising  the  stom-  A      ^  ^  \S* 

ach  contents  and  paralysing  r^  w  s 

the  intestine  with   opium.        Jjjjj®  ^^  ^ 

This  occurs  in  a  small  pro- 

portion of  the  animals  ex-  t  /)<•/*  ^1)  V  '!/ 

perimented  on,  and  the  con-      ^     ^ 

tents  of  the  intestine,  unlike 

what  was  found  in  the  case 

of    the    cholera    organism,       ^  i£y/~  I    if 

were  turbid  in  appearance,  ^\\\/"^ 

and  had  a  markedly  foetid 

odour.     When  tested  by  in- 

traperitoneal   injection,   its  "*Xr^-* 

effects  are  somewhat  of  the  *^  ~-f  j 

same  nature  as  those  of  the 

cholera   organism,    but    its 

virulence  £  of  a  much  lower    FlG'  Hl.-Fmkler  and  Prior  s  spirillum  ;  from 

orcjer  an   agar   culture   of    twenty  -  four    hours 

Stained  with  carbol-fuchsin.      x  1  000. 


from  the  cavity  of  a  decayed 

tooth  in  a  human  subject  is  almost  certainly  the  same  organism  as 

Finkler  and  Prior's  spirillum. 

Deneke's  Spirillum.  —  This  organism  was  obtained  from  old  cheese,  and 
is  also  known  as  the  spirillum  tyrogenum.  It  closely  resembles  Koch's 
spirillum  in  microscopic  appearances,  though  it  is  rather  thinner  and 
smaller  Its  growth  in  gelatin  is  also  somewhat  similar,  but  liquefaction 
proceeds  more  rapidly,  and  the  bell-shaped  depression  on  the  surface  is 
larger  and  shallower,  whilst  the  growth  has  a  more  distinctly  yellowish 
tint.  The  colonies  in  plates  also  show  points  of  resemblance,  though  the 
youngest  colonies  are  rather  smoother  and  more  regular  on  the  surface,  and 
liquefaction  occurs  more  rapidly  than  in  the  case  of  the  cholera  organism. 
The  colonies  have,  on  naked-eye  examination,  a  distinctly  yellowish  col  our. 
The  organism  does  not  give  the  cholera-red  reaction,  and  on  potato  it 
forms  a  thin  yellowish  layer  when  incubated  above  30°  C.  When  tested 
by  intraperitoneal  injection  and  by  other  methods  it  is  found  to  possess 
very  feeble,  or  almost  no,  pathogenic  properties.  Koch  found  that  this 
organism,  when  administered  through  the  stomach  in  the  same  way  as 
the  cholera  organism,  produced  a  fatal  result  in  three  cases  out  of 
fifteen.  Deneke's  spirillum  is  usually  regarded  as  a  comparatively 
harmless  saprophyte. 


CHAPTER  XVIII. 

INFLUENZA,  PLAGUE,  RELAPSING  FEVER, 
MALTA  FEVER,  YELLOW  FEVER. 

INFLUENZA. 

THE  first  accounts  of  the  organism  now  known  as  the  influenza 
bacillus  were  published  simultaneously  by  Pfeiffer,  Kitasato,  and 
Canon,  in  January  1892.  The  two  first-mentioned  observers 

found  it  in  the  bronchial 

-  \r''jt    *  <?£     ^  ,  sputum,  and  obtained  pure 

.  V  cultures,   and    Canon    ob- 

.*         ',''***,  f-vv  ?'  served  it  in  the  blood  in  a 

*'  A  *     v       few  cases  of  the  disease. 

.  *   »f  *  £$&>•  "     ,    •'         'i  9      It  is,  however,  to  Pfeiffer 's 

»,1  '.'-*      *•**     -        ,  .        work  that  we  owe  most  of 

*    i v/ •>'/''('     e  •*       '•?*"         our  knowledge  regarding 

*c'*~ l  '    *?-X.^»    *'  •      /     'ts  characters  and  action. 

^     ./    y*  '  m     His    results     have     been 

'  *  .  ,  ;       amply  confirmed  by  those 

*•    \  f      /T       ,~          .  "\     x",  of  others  in  various  epi- 

Ai  -/          '.  *J^     .',  V    ,  demies  of  the  disease,  and 

V'  V  V  v*  ,.  this 


x      *i  v  " .  ^ '  generally  accepted  as  the 

cause  of  the  disease,   al- 

FIG.  142. -Influenzabacim  from  a  culture     though   absolute    proof    is 

Stained  with  carbol-fuchsin.      x  1000.        sti11  wanting. 

Microscopical  Char- 
acters.— The  influenza  bacilli  as  seen  in  the  sputum  are  very 
minute  rods  not  exceeding  1  '5  /x  in  length  and  *3  p  in  thickness. 
They  are  straight,  with  rounded  ends,  and  sometimes  stain  more 
deeply  at  the  extremities  (Fig.  142).  The  bacilli  occur  singly 
or  form  clumps  by  their  aggregation,  but  do  not  grow  into  chains. 
They  show  no  capsule.  They  take  up  the  basic  aniline  stains 

420 


CULTIVATION   OF   BACILLI  421 

somewhat  feebly,  and  are  best  stained  by  a  weak  solution  (1  : 10) 
of  carbol-fuchsin  applied  for  5  to  10  minutes.  They  lose  the 
stain  in  Gram's  method.  They  are  non-motile,  and  do  not  form 
spores. 

In  many  cases  of  the  disease,  especially  in  the  early  stages  of 
the  more  acute,  influenza  bacilli  are  present  in  large  numbers 
and  may  be  easily  found.  On  the  other  hand,  it  is  often 
difficult  or  impossible  to  find  them,  even  when  the  symptoms 
are  severe ;  this  may  be  due  to  the  restriction  of  the  organisms 
to  some  part  not  readily  accessible,  or  it  may  be  that  they 
actually  die  out  in  great  part  while  the  effects  of  their  toxins 
persist.  It  has  also  been  observed  in  recent  epidemics,  in  which 
the  disease  has  been  less  widespread  and  on  the  whole  less 
severe,  that  the  period  during  which  the  bacilli  have  been  readily 
demonstrable  in  the  secretions  has  been  on  the  average  shorter 
than  in  the  previous  epidemics. 

Cultivation. — The  best  medium  for  the  growth  of  the 
influenza  bacillus  is  blood  agar  (see  page  38),  which  was  intro- 
duced by  Pfeiffer  for  this  purpose.  He  obtained  growths  of  the 
bacilli  on  agar  which  had  been  smeared  with  influenza  sputum, 
but  he  failed  to  get  any  sw6-cultures  on  the  agar  media  or  on 
serum.  The  growth  in  the  first  cultures  he  considered  to  be 
probably  due  to  the  presence  of  certain  organic  substances  in 
the  sputum,  and  accordingly  he  tried  the  expedient  of  smearing 
the  agar  with  drops  of  blood  before  making  the  inoculations. 
In  this  way  he  completely  succeeded  ,in  attaining  his  object. 
The  blood  of  the  lower  animals  is  suitable,  as  well  as  human 
blood ;  and  the  favouring  influences  of  the  blood  would  appear 
to  be  due  to  the  haemoglobin,  as  a  solution  of  this  substance  is 
equally  effective.  The  colonies  of  the  influenza  bacilli  on  blood 
agar,  incubated  at  37°  C.,  appear  within  twenty-four  hours,  in 
the  form  of  minute  circular  dots  almost  transparent,  like  drops 
of  dew.  When  numerous,  the  colonies  are  scarcely  visible  to 
the  naked  eye,  but  when  sparsely  arranged  they  may  reach  the 
size  of  a  small  pin's  head.  This  size  is  generally  reached  on  the 
second  day.  The  bacilli  die  out  somewhat  quickly  in  cultures, 
and  in  order  to  keep  them  alive  sub-cultures  should  be  made 
every  four  or  five  days.  By  this  method  the  cultures  may  be 
maintained  for  an  indefinite  period.  Growth  on  the  ordinary 
agar  media  is  slight  and  somewhat  uncertain  ;  there  is,  however, 
evidence  that  growth  is  more  marked  when  other  organisms  are 
present,  that  is,  is  favoured  by  symbiosis.  Neisser,  for  example, 
was  able  to  cultivate  the  influenza  bacillus  on  plain  agar 
through  several  generations  by  growing  the  xerosis  bacillus 


422  INFLUENZA 

along  with  it ;  dead  cultures  of  the  latter  had  not  the  same 
favouring  effect.  A  very  small  amount  of  growth  takes  place 
in  bouillon,  but  it  is  more  marked  when  a  little  fresh  blood  is 
added.  The  growth  forms  a  thin  whitish  deposit  at  the  bottom 
of  the  flask.  The  limits  of  growth  are  from  25°  to  42°  C.,  the 
optimum  temperature  being  that  of  the  body.  The  influenza 
bacillus  is  a  strictly  aerobic  organism. 

The  powers  of  resistance  of  this  organism  are  of  a  low  order. 
Pfeiffer  found  that  dried  cultures  kept  at  the  ordinary  tempera- 
ture were  usually  dead  in  twenty  hours,  and  that  if  sputum  were 
kept  in  a  dry  condition  for  two  days,  all  the  influenza  bacilli 
were  dead,  or  rather,  cultures  could  be  no  longer  obtained. 
Their  duration  of  life  in  ordinary  water  is  also  short,  the  bacilli 
usually  being  dead  within  two  days.  From  these  experiments 
Pfeiffer  concludes  that  outside  the  body  in  ordinary  conditions 
they  cannot  multiply,  and  can  remain  alive  only  for  a  short  time. 
The  mode  of  infection  in  the  disease  he  accordingly  considers  to 
be  chiefly  by  means  of  fine  particles  of  disseminated  sputum,  etc. 
Distribution  in  the  Body. — The  bacilli  are  found  chiefly  in 
the  respiratory  passages  in  influenza.  They  may  be  present  in 
large  numbers  in  the  nasal  secretion,  generally  mixed  with  a 
considerable  number  of  other  organisms,  but  it  is  in  the  small 
masses  of  greenish-yellow  sputum  from  the  bronchi  that  they 
occur  in  largest  numbers,  and  in  many  cases  almost  in  a  state 
of  purity.  They  occur  in  clumps  which  may  contain  as  many 
as  100  bacilli,  and  in  the  early  stages  of  the  disease  are  chiefly 
lying  free.  As  the  disease  advances,  they  may  be  found  in 
considerable  numbers  within  the  leucocytes,  and  towards  the 
end  of  the  disease  a  large  proportion  have  this  position.  It  is 
a  matter  of  considerable  importance,  however,  that  they  may 
persist  for  weeks  after  symptoms  of  the  disease  have  disappeared, 
and  may  still  be  detected  in  the  sputum.  Especially  is  this  the 
case  when  there  is  any  chronic  pulmonary  disease.  They  also 
occur  in  large  numbers  in  the  capillary  bronchitis  and  catarrhal 
pneumonia  of  influenza,  as  Pfeiffer  showed  by  means  of  sections 
of  the  affected  parts.  In  these  sections  he  found  the  bacilli 
lying  amongst  the  leucocytes  which  filled  the  minute  bronchi, 
and  also  penetrating  between  the  epithelial  cells  and  into  the 
superficial  parts  of  the  mucous  membrane.  Other  organisms 
also,  especially  Fraenkel's  pneumococcus,  may  be  concerned  in 
the  pneumonic  conditions  following  influenza.  In  some  cases 
influenza  occurs  in  tubercular  subjects,  or  is  followed  by  tubercular 
affection,  in  which  cases  both  influenza  and  tubercle  bacilli  may 
be  found  in  the  sputum.  In  such  a  condition  the  prognosis  is 


DISTRIBUTION   OF   BACILLI  423 

very  grave.  Regarding  the  presence  of  influenza  bacilli  in  the 
other  pulmonary  complications  following  influenza,  much  in- 
formation is  still  required.  Occasionally  in  the  foci  of  sup- 
purative  softening  in  the  lung  the  influenza  bacilli  have  been 
found  in  a  practically  pure  condition.  In  cases  of  empyema  the 
organisms  present  would  appear  to  be  chiefly  streptococci  and 
pneumococci;  whilst  in  the  gangrenous  conditions,  which 
sometimes  occur,  a  great  variety  of  organisms  has  been  found. 

Pfeiffer's  observations  on  a  large  series  of  cases  convinced 
him  that  the  organism  was  very  rarely  present  in  the  blood — 
that  in  fact  its  occurrence  there  must  be  looked  upon  as 
exceptional.  The  conclusions  of  other  observers  have,  on  the 
whole,  confirmed  this  statement,  and  it  is  probable  that  the 
chief  symptoms  in  the  disease  are  due  to  toxins  absorbed  from 
the  respiratory  tract  (vide  infra).  The  bacillus  may  be  present 
in  some  of  the  lesions  complicating  influenza.  Pfeiffer  found 
it  in  inflammation  of  the  middle  ear,  but  in  a  case  of  meningitis 
following  influenza  Fraenkel's  diplococcus  was  present.  In  a 
few  cases  of  meningitis,  however,  the  influenza  bacillus  has  been 
found,  sometimes  alone,  sometimes  along  with  pyogenic  cocci 
(Pfuhl  and  Walter,  Cornil  and  Durante) ;  Pfuhl  considers  that 
in  these  the  path  of  infection  is  usually  a  direct  one  through 
the  roof  of  the  nasal  cavity.  This  observer  also  found  post 
mortem,  in  a  rapidly  fatal  case  with  profound  general  symptoms, 
influenza  bacilli  in  various  organs,  both  within  and  outside  of 
the  vessels.  In  a  few  cases  also  the  bacilli  have  been  found  in 
the  brain  and  its  membranes  with  little  tissue  change  in  the 
parts  around. 

Extensive  observations  on  the  bacteriology  of  the  respiratory 
system  show  that  influenza-like  bacilli  may  be  present  in  a  great 
variety  of  conditions ;  we  have,  in  fact,  once  more  to  do  with  a 
group  of  organisms  with  closely  allied  characters,  of  which 
Pfeiffer's  influenza  bacillus  was  the  first  recognised  example. 
These  "pseudo-influenza"  bacilli  have  been  obtained  from  the 
fauces,  bronchi,  and  lungs  in  inflammatory  conditions,  and  also  in 
various  specific  fevers.  To  this  group  belongs  the  bacillus  which 
has  been  cultivated  from  cases  of  whooping-cough  by  Spengler, 
Jochmann,  Davis,  and  others,  and  which  is  present  in  considerable 
numbers  in  a  large  proportion  of  cases  of  this  disease.  Wollstein 
has  obtained  a  marked  agglutinative  reaction  on  this  organism 
by  the  serum  of  whooping-cough  patients,  all  the  sera  examined 
giving  a  positive  reaction  in  a  dilution  of  at  least  1  : 100  on  all 
the  strains  of  the  organism  isolated  ;  on  the  other  hand,  clumping 
was  never  obtained  with  a  normal  serum  in  a  greater  dilution 


424  INFLUENZA 

than  1:10.  Davis,  by  inoculation  of  the  fauces  of  a  healthy  man 
with  this  organism,  produced  inflammatory  change  with  febrile 
reaction,  but  not  the  characteristic  symptoms  of  whooping-cough. 
There  is  presumptive  evidence  in  favour  of  the  bacillus  being 
etiologically  related  to  the  disease,  though  the  matter  cannot  yet 
be  considered  settled.  M  tiller's  "trachoma  bacillus"  (p.  192)  is 
a  member  of  the  same  group.  All  these  organisms  are  very 
restricted  in  their  growth,  and  require  the  addition  of  blood  or 
haemoglobin  to  the  ordinary  culture  media ;  hence  they  are  some- 
times spoken  of  as  hsemophilic  bacteria.  Some  of  the  examples 
are  a  little  larger  than  the  influenza-bacillus,  and  tend  to  form 
short  filaments,  but  others  are  quite  indistinguishable.  All  of 
them  also  seem  to  have  very  feeble  pathogenic  properties  towards 
the  lower  animals.  At  present  it  can  scarcely  be  claimed  as 
possible  to  identify  Pfeiffer's  bacillus  by  its  microscopic  and 
cultural  characters. 

Experimental  Inoculation. — There  is  no  satisfactory  evidence 
that  any  of  the  lower  animals  suffer  from  influenza  in  natural 
conditions,  and  accordingly  we  cannot  look  for  very  definite 
results  from  experimental  inoculation.  Pfeiffer,  by  injecting 
living  cultures  of  the  organism  into  the  lungs  of  monkeys,  in 
three  cases  produced  a  condition  of  fever  of  a  remittent  type. 
There  was,  however,  little  evidence  that  the  bacilli  had  under- 
gone multiplication,  the  symptoms  being  apparently  produced 
by  their  toxins.  In  the  case  of  rabbits,  intravenous  injection  of 
living  cultures  produces  dyspnoea,  muscular  weakness,  and 
slight  rise  of  temperature,  but  the  bacilli  rapidly  disappear  in 
the  body,  and  exactly  similar  symptoms  are  produced  by 
injection  of  cultures  killed  by  the  vapour  of  chloroform. 
Pfeiffer,  therefore,  came  to  the  conclusion  that  the  influenza 
bacilli  contain  toxic  substances  which  can  produce  in  animals 
some  of  the  substances  of  the  disease,  but  that  animals  are  not 
liable  to  infection,  the  bacilli  not  having  power  of  multiplying 
to  any  extent  in  their  tissues. 

Cantani  succeeded  in  producing  infection  to  some  extent  in 
rabbits,  by  injecting  the  bacilli  directly  into  the  anterior 
portion  of  the  brain.  In  these  experiments  the  organisms 
spread  to  the  ventricles,  and  then  through  the  spinal  cord  by 
means  of  the  central  canal,  afterwards  infecting  the  substance 
of  the  cord.  An  acute  encephalitis  was  thus  produced,  and 
sometimes  a  purulent  condition  in  the  lateral  ventricles.  The 
bacilli  were,  however,  never  found  in  the  blood  or  in  other  organs. 
Similar  symptoms  were  also  produced  by  injection  of  dead 
cultures,  though  in  this  case  the  dose  required  to  be  five  or  six 


METHODS   OF   EXAMINATION  425 

times  larger.  Cantani  therefore  concludes  that  the  brain 
substance  is  the  most  suitable  nidus  for  their  growth,  but 
agrees  with  Pfeiffer  in  believing  that  the  chief  symptoms  are 
produced  by  toxins  resident  in  the  bodies  of  the  bacilli.  He 
made  control  experiments  by  injecting  other  organisms,  and  also 
by  injecting  inert  substances  into  the  cerebral  tissue. 

The  evidence,  accordingly,  that  the  influenza  bacillus  is  the 
cause  of  the  disease  rests  chiefly  on  the  well-established  fact  that 
it  is  always  present  in  the  secretions  of  the  respiratory  tract  in 
true  cases  of  influenza,  and  often  in  very  large  numbers.  The 
observed  relationships  of  the  organism  to  lesions  in  the  lungs 
and  elsewhere  leave  no  room  for  doubt  that  it  is  possessed  of 
pathogenic  properties,  but  we  cannot  yet  maintain  that  its  causal 
relationship  to  epidemic  influenza  is  absolutely  established.  A 
certain  amount  of  confirmatory  evidence  has  been  supplied  by 
the  results  of  experiment. 

Methods  of  Examination — (a)  Microscopic. — A  portion  of  the 
greenish-yellow  purulent  material  which  often  occurs  in  little 
round  masses  in  the  sputum  should  be  selected,  and  film  prepara- 
tions should  be  made  in  the  usual  way.  Films  are  best  stained 
by  Ziehl-Neelsen  carbol-f  uchsin  diluted  with  ten  parts  of  water, 
the  films  being  stained  for  ten  minutes  at  least.  In  sections  of 
the  tissues,  such  as  the  lungs,  the  bacilli  are  best  brought  out, 
according  to  Pfeiffer,  by  staining  with  the  same  solution  as  above 
for  half  an  hour.  The  sections  are  then  placed  in  alcohol 
containing  a  few  drops  of  acetic  acid,  in  which  they  are 
dehydrated  and  slightly  decolorised  at  the  same  time.  They 
should  be  allowed  to  remain  till  they  have  a  moderately  light 
colour,  the  time  varying  according  to  their  appearance.  They 
are  then  washed  in  pure  alcohol,  cleared  in  xylol,  and  afterwards 
mounted  in  balsam. 

(b)  Cultures. — A  suitable  portion  of  the  greenish -yellow 
material  having  been  selected  from  the  sputum,  it  should  be 
washed  well  in  several  changes  of  sterilised  water.  A  portion 
should  then  be  taken  on  a  platinum  needle,  and  successive 
strokes  made  on  the  surface  of  blood-agar  tubes.  The  tubes 
should  then  be  incubated  at  37°  C.,  when  the  transparent 
colonies  of  the  influenza  bacillus  will  appear,  usually  within 
twenty-four  hours.  These  should  give  a  negative  result  on 
inoculation  on  ordinary  agar  media. 

PLAGUE. 

The  bacillus  of  oriental  plague  or  bubonic  pest  was  discovered 
independently  by  Kitasato  and  Yersin  during  the  epidemic  at 


426 


PLAGUE 


Hong  Kong  in  1894.  The  results  of  their  investigations,  which 
were  published  almost  at  the  same  time,  agree  in  most  of  the 
important  points.  They  cultivated  the  same  organism  from  a 
large  number  of  cases  of  plague,  and  reproduced  the  disease  in 
susceptible  animals  by  inoculation  of  pure  cultures.  It  is  to 
be  noted  that  during  an  epidemic  of  plague,  sometimes  even 
preceding  it,  a  high  mortality  has  been  observed  amongst  certain 


FIG.  143. — Film  preparation  from  a  plague  bubo  showing  enormous  numbers 

of  bacilli,  most  of  which  show  well-marked  bipolar  staining. 

Stained  with  weak  gentian-violet,      x  1000. 

animals,  especially  rats  and  mice,  and  that  from  the  bodies  of 
these  animals  found  dead  in  the  plague-stricken  district,  the 
same  bacillus  was  obtained  by  Kitasato  and  also  by  Yersin. 

Bacillus  of  Plague — Microscopical  Characters. — As  seen  in 
the  affected  glands  or  buboes  in  this  disease,  the  bacilli  are 
small  oval  rods,  somewhat  shorter  than  the  typhoid  bacillus, 
and  about  the  same  thickness  (Fig.  143),  though  considerable 
variations  in  size  occur.  They  have  rounded  ends,  and  in 
stained  preparations  a  portion  in  the  middle  of  the  bacillus  is 


BACILLUS   OF   PLAGUE 


427 


often  left  uncoloured,  giving  the  so-called  "  polar  staining."     In 

films  from  the  tissues  they 

are     found     scattered 

amongst  the  cells,  for  the 

most    part    lying   singly, 

though  pairs  are  also  seen. 

On  the  other  hand,  in  cul- 
tures in  fluids,  e.g.  bouillon, 

they  grow  chiefly  in  chains, 

sometimes  of  considerable 

length,   the   form   known 

as  a  streptobacillus  result- 
ing (Fig.  145).     In  young 

agar   cultures   the    bacilli 

show  greater  variation  in 

size,  and  polar  staining  is 

less   marked   than  in  the 

tissues:    sometimes   forms    Fia   14  4. -Bacillus  of  plague  from  a  young 
,.  .  j       111          i.  culture  on  agar. 

ot  considerable  length  are   staine(T  with  weak  carbol-fuchsin.     x  1000. 

present.     After  a  time  in- 
volution forms  appear,  especially  when  the  surface  of  the  agar 

is  dry  ;  but  the  formation 
of  these  is  much  more 
rapid  and  more  marked 
when  2-5  per  cent  of 
sodium  chloride  is  added 
to  the  medium,  constitut- 
ing the  so-called  "salt- 
agar  "  (Hankin  and  Leu- 
mann).  On  this  medium, 
especially  with  the  higher 
percentage,  the  involution 
forms  assume  a  great  size 
and  a  striking  variety  of 
shapes,  large  globular, 
oval,  or  pyriform  bodies 
resulting  (Fig.  146) ;  with 

FIG.  145.— Bacillus  of  plague  iii  chains  show-  about  2  per  cent  sodium 
ing  polar  staining.  From  a  young  culture  chloride, aftertwenty-f our 
in  bouillon.  ,  '.  ,  , .  ,1 

Stained  with  thionin-blue.      x  1000.  hours      incubation,      the 

most  striking  feature  is  a 

general  enlargement  of  all  the  bacilli.     Sometimes  in  the  tissues 

they  are  seen  to  be  surrounded  by  an  unstained  capsule,  though 

this  appearance  is  by  no  means  common.     They  do  not  form 


428  PLAGUE 

spores.  Gordon,  who  has  found  that  they  possess 
which,  however,  stain  with  difficulty,  states  that  they  are 
motile.  Most  observers,  however,  and  with  these  we  agree, 
have  failed  to  find  evidence  of  true  motility.  They  stain  readily 
with  the  basic  aniline  stains,  but  are  decolorised  by  Gram's 
method. 

Cultivation. — From  the  affected  glands,  etc.,  the  bacillus  can 
readily  be  cultivated  on  the  ordinary  media.  It  grows  best  at 
the  temperature  of  the  body,  though  growth  occurs  as  low  as 
18°  C.  On  agar  and  on  blood  serum  the  colonies  are  whitish 

circular  discs  of  somewhat 
transparent  appearance 
and  smooth,  shining  sur- 
face. When  examined 
with  a  lens,  their  borders 
appear  slightly  wavy.  In 
stroke  cultures  on  agar 
there  forms  a  continuous 
line  of  growth  with  the 
same  appearance,  showing 
partly  separated  colonies 
,  at  its  margins.  When 

agar  cultures  are  kept  at 
the  room  temperature, 
some  of  the  colonies  may 
show  a  more  luxuriant 

growth  with  more  opaque 
FIG.  146.— Culture  of  the  bacillus  of  plague    & 

on  4  per  cent  salt  agar,  showing  involution   appearance  than  the   rest 

forms  of  great  variety  of  size  and  shape.       of  the  growth,  the  appear- 

Stained  with  carbol-thionin-blue.      x  1000.      ance  in  fact  being  often 

such   as    to   suggest  the 

presence  of  impurities  in  the  cultures.  In  stab  cultures  in 
peptone  gelatin,  growth  takes  place  along  the  needle  track  as 
a  white  line,  composed  of  small  spherical  colonies.  On  the 
surface  of  the  gelatin  a  thin,  semi-transparent  layer  may  be 
formed,  which  is  usually  restricted  to  the  region  of  puncture, 
though  sometimes  it  may  spread  to  the  wall  of  the  tube  ;  some- 
times, however,  there  is  practically  no  surface  growth.  There  is 
no  liquefaction  of  the  medium.  In  gelatin  plates  the  superficial 
colonies  develop  first  and  form  slightly  raised  semi-transparent 
discs  with  somewhat  crenated  margins ;  the  deeper  colonies  are 
smaller  and  of  spherical  shape  with  smooth  outline.  In  bouillon 
the  growth  usually  forms  a  slightly  granular  or  powdery  deposit 
at  the  foot  and  sides  of  the  flask,  somewhat  resembling  that  of 


DISTRIBUTION   OF   BACILLI  429 

a  streptococcus.  If  oil  or  melted  butter  is  added  to  the  bouillon 
so  that  drops  float  on  the  surface,  then  a  striking  mode  of  growth 
may  result,  to  which  the  term  "  stalactite "  has  been  applied. 
This  consists  in  the  growth  starting  from  the  under  surface  of 
the  fat  globules  and  extending  downwards  in  the  form  of 
pendulous,  string-like  masses.  These  masses  are  exceedingly 
delicate,  and  readily  break  off  on  the  slightest  shaking  of  the 
flask  ;  accordingly  during  their  formation  the  culture  must  be 
kept  absolutely  at  rest.  This  manner  of  growth  constitutes  an 
important  but  not  absolutely  specific  character  of  the  organism  ; 
unfortunately  it  is  not  supplied  by  all  races  of  the  organism,  and 
varies  from  time  to  time  with  the  same  race.  The  organism 
flourishes  best  in  an  abundant  supply  of  oxygen ;  in  strictly 
anaerobic  conditions  almost  no  growth  takes  place. 

The  organism  in  its  powers  of  resistance  corresponds  with 
other  spore-free  bacilli,  and  is  readily  killed  by  heat,  an  exposure 
for  an  hour  at  58°  C.  being  fatal.  On  the  other  hand  it  has 
remarkable  powers  of  resistance  against  cold  ;  it  has  been  exposed 
to  a  temperature  several  degrees  below  freezing-point  without 
being  killed.  Experiments  on  the  effects  of  drying  have  given 
somewhat  diverse  results,  but  as  a  rule  the  organism  has  been 
found  to  be  dead  after  being  dried  for  from  six  to  eight  days, 
though  sometimes  it  has  survived  the  process  for  a  longer  period  ; 
exposure  to  direct  sunlight  for  three  or  four  hours  kills  it.  When 
cultivated  outside  the  body  the  organism  often  loses  its  virulence, 
but  some  races  remain  virulent  in  cultures  for  a  long  period  of 
time. 

Anatomical  Changes  and  Distribution  of  Bacilli. — The 
disease  occurs  in  several  forms,  the  bubonic  and  the  pulmonary 
being  the  best  recognised  ;  to  these  may  be  added  the  septiccemic. 
The  most  striking  feature  in  the  bubonic  form  is  the  affection  of 
the  lymphatic  glands,  which  undergo  intense  inflammatory 
swelling,  attended  with  haemorrhage,  and  generally  ending  in 
a  greater  or  less  degree  of  necrotic  softening  if  the  patient  lives 
long  enough.  The  connective  tissue  around  the  glands  is 
similarly  affected.  The  bubo  is  thus  usually  formed  by  a 
collection  of  enlarged  glands  fused  by  the  inflammatory  swelling. 
True  suppuration  is  rare.  Usually  one  group  of  glands  is 
affected  first,  constituting  the  primary  bubo — in  the  great 
majority  the  inguinal  or  the  axillary  glands — and  afterwards 
other  groups  may  become  involved,  though  to  a  much  less 
extent.  Along  with  these  changes  there  is  great  swelling  of 
the  spleen,  and  often  intense  cloudy  swelling  of  the  cells  of  the 
kidneys,  liver,  and  other  organs.  There  may  also  occur  secondary 


430  PLAGUE 

areas  of  haemorrhage  and  necrosis,  chiefly  in  the  lungs,  liver, 
and  spleen.  The  bacilli  occur  in  enormous  numbers  in  the 
swollen  glands,  being  often  so  numerous  that  a  film  preparation 
made  from  a  scraping  almost  resembles  a  pure  culture  (Fig. 
143).  In  sections  of  the  glands  in  the  earlier  stages  the  bacilli 
are  found  to  form  dense  masses  in  the  lymph  paths  and  sinuses 


FIG.  147. — Section  of  a  human  lymphatic  gland  in  plague,  showing  the 

injection  of  the  lymph  paths  and  sinuses  with  masses  of  plague  bacilli — seen 
as  black  areas. 

Stained  with  carbol-thioniu-blue.      x  50. 

(Fig.  147),  often  forming  an  injection  of  them;  they  may  also 
be  seen  growing  as  a  fine  reticulum  between  the  cells  of  the 
lymphoid  tissue.  At  a  later  period,  when  disorganisation  of 
the  gland  has  occurred,  they  become  irregularly  mixed  with  the 
cellular  elements.  Later  still  they  gradually  disappear,  and 
when  necrosis  is  well  advanced  it  may  be  impossible  to  find  any 
— a  point  of  importance  in  connection  with  diagnosis.  In  the 
spleen  they  may  be  very  numerous  or  they  may  be  scanty, 
according  to  the  amount  of  blood  infection  which  has  occurred  ; 


EXPERIMENTAL   INOCULATION  431 

in  the  secondary  lesions  mentioned  they  are  often  abundant. 
In  the  pulmonary  form  the  lesion  is  the  well-recognised  "  plague 
pneumonia."  This  is  of  broncho-pneumonic  type,  though  large 
areas  may  be  formed  by  confluence  of  the  consolidated  patches, 
and  the  inflammatory  process  is  attended  usually  by  much 
haemorrhage ;  the  bronchial  glands  show  inflammatory  swelling. 
Clinically  there  is  usually  a  fairly  abundant  frothy  sputum  often 
tinted  with  blood,  and  in  it  the  bacilli  may  be  found  in  large 
numbers.  Sometimes,  however,  cough  and  expectoration  may 
be  absent.  The  disease  in  this  form  is  said  to  be  invariably 
fatal.  In  the  septiccemic  form  proper  there  is  no  primary  bubo 
discoverable,  though  there  is  almost  always  general  enlargement 
of  lymphatic  glands ;  here  also  the  disease  is  of  specially  grave 
character.  A  bubonic  case  may,  however,  terminate  with  septi- 
caemia ;  in  fact  all  intermediate  forms  occur.  An  intestinal  form 
with  extensive  affection  of  the  mesenteric  glands  has  been 
described,  but  it  is  exceedingly  rare — so  much  so  that  many 
observers  with  extensive  experience  have  doubted  its  occurrence. 
In  the  various  forms  of  the  disease  the  bacilli  occur  also  in  the 
blood,  in  which  they  may  be  found  during  life  by  microscopic 
examination,  chiefly,  however,  just  before  death  in  very  severe 
and  rapidly  fatal  cases.  The  examination  of  the  blood  by  means 
of  cultivation  experiments  is,  however,  a  much  more  reliable 
procedure.  For  this  purpose  about  1  c.c.  of  blood  may  be  with- 
drawn from  a  vein  and  distributed  in  flasks  of  bouillon  (p.  68). 
It  may  be  said  from  the  results  of  different  investigators  that 
the  bacillus  may  be  obtained  by  culture  in  fully  50  per  cent  of 
the  cases,  though  the  number  will  necessarily  vary  in  different 
epidemics.  The  Advisory  Committee,  recently  appointed,  found 
that  in  some  septicaemic  cases  the  bacilli  may  be  present  in  the 
blood  in  large  numbers  two,  or  even  three,  days  before  death, 
though  this  is  exceptional. 

The  above  types  of  the  disease  are  usually  classified  together 
under  the  heading  pestis  major,  but  there  also  occur  mild  forms 
to  which  the  term  pestis  minor  is  applied.  In  these  latter  there 
may  be  a  moderate  degree  of  swelling  of  a  group  of  glands, 
attended  with  some  pyrexia  and  general  malaise,  or  there  may 
be  little  more  than  slight  discomfort.  Between  such  and  the 
graver  types,  cases  of  all  degrees  of  severity  are  met  with. 

Experimental  Inoculation. — Mice,  guinea-pigs,  rats,  and 
rabbits  are  susceptible  to  inoculation,  the  two  former  being  on 
the  whole  most  suitable  for  experimental  purposes.  After  sub- 
cutaneous injection  there  occurs  a  local  inflammatory  cedema, 
which  is  followed  by  inflammatory  swelling  of  the  corresponding 


432 


PLAGUE 


lymphatic  glands,  and  thereafter  by  a  general  infection.  The 
lesions  in  the  lymphatic  glands  correspond  in  their  main 
characters  with  those  in  the  human  subject,  although  usually 
at  the  time  of  death  they  have  not  reached  a  stage  so  advanced. 
By  this  method  of  inoculation  mice  usually  die  in  1-3  days, 
guinea-pigs  and  rats  in  2-5  days,  and  rabbits  in  4-7  days. 
Post  mortem  the  chief  changes,  in  addition  to  the  glandular 
enlargement,  are  congestion  of  internal  organs,  sometimes  with 
haemorrhages,  and  enlargement  of  the  spleen  ;  the  bacilli  are 
numerous  in  the  lymphatic  glands  and  usually  in  the  sjpleen 

(Fig.  148),  and  also, 
though  in  somewhat  less 
degree,  throughout  the 
blood.  Infection  can 
also  be  produced  by 
smearing  the  material 

on  *^e  conjunctiva  °r 

mucous    membrane     of 
thenose,andthismethod 
of  inoculation  has  been 
successfully   applied   in 
cases  where  the  plague 
bacilli  are  present  along 
with      other      virulent 
organisms,  e.g.  in  sputum 
along  with  pneumococci. 
FIG.  148.—  Film  preparation  of  spleen  of  rat    Rats  and  mice  can  also 
after  inoculation  with  the  bacillus  of  plague,    be  infected    by  feeding 
showing  numerous  bacilli,  most  of  which   either  with  pure  cultures 
are  somewhat  plump.  .,,       .  ,, 

Stained  with  carbol-thionin-blue.      x  1000.          or  ^^  Pieces  of  organs 

from  cases  of  the  disease, 

though  in  this  case  infection  probably  takes  place  through  the 
mucous  membrane  of  the  mouth  and  adjacent  parts,  and  only 
to  a  limited  extent,  if  at  all,  by  the  alimentary  canal.  Monkeys 
also  are  highly  susceptible  to  infection,  and  it  has  been  shown 
in  the  case  of  these  animals  that  when  inoculation  is  made  on 
the  skin  surface,  for  example,  by  means  of  a  spine  charged  with 
the  bacillus,  the  glands  in  relation  to  the  part  may  show  the 
characteristic  lesion  and  a  fatal  result  may  follow  without  there 
being  any  noticeable  lesion  at  the  primary  seat.  This  fact 
throws  important  light  on  infection  by  the  skin  in  the  human 
subject.  The  disease  may  also  extensively  affect  monkeys  by 
natural  means  during  an  epidemic. 

Paths  and  Mode  of  Infection.  —  Plague  bacilli  may  enter 


PATHS   AND   MODE   OF   INFECTION          433 

the  system  by  the  skin  surface  through  small  wounds,  cracks, 
abrasions,  etc.,  and  in  such  cases  there  is  usually  no  reaction 
at  the  site  of  entrance.  This  last  fact  is  in  accordance  with 
what  has  been  stated  above  with  regard  to  experiments  on 
monkeys.  The  path  of  infection  is  shown  by  the  primary 
buboes,  which  are  usually  in  the  glands  through  which  the 
skin  is  drained,  those  in  the  groin  being  the  commonest  site. 
Absolute  proof  of  the  possibility  of  infection  by  the  skin  is 
supplied  by  several  cases  in  which  the  disease  has  been  acquired 
at  post-mortem  examinations,  the  lesions  of  the  skin  surface 
being  in  the  majority  of  these  of  trifling  nature ;  in  only  two 
was  there  local  reaction  at  the  site  of  inoculation.  In  most  of 
these  cases  the  period  of  incubation  has  been  from  two  to  three 
days ;  under  natural  conditions  of  infection  the  average  period 
is  within  five  days.  While  infection  may  occur  by  accidental 
inoculation  through  small  wounds  of  the  skin  surface,  it  appears 
in  the  majority  of  cases  to  take  place  by  means  of  the  bites 
of  fleas.  For  some  time  it  had  been  known  that  plague  bacilli 
might  be  found  for  some  time  afterwards  in  the  stomach  of  fleas 
allowed  to  feed  on  animals  suffering  from  plague,  and  some 
observers,  for  example  Simond,  had  succeeded  in  transmitting 
the  disease  to  other  animals  by  means  of  the  infected  insects. 
Most  observers,  however,  had  obtained  negative  results,  and  it 
was  only  by  the  work  of  the  Advisory  Committee  appointed  by 
the  Secretary  of  State  for  India  in  19051  that  the  importance 
of  this  means  of  infection  was  established.  By  carefully  planned 
experiments  the  Committee  showed  that  the  disease  could  be 
transmitted  from  a  plague  rat  to  a  healthy  rat  kept  in  adjacent 
cages  when  fleas  were  present ;  whereas  this  did  not  occur  when 
means  were  taken  to  prevent  the  access  of  fleas,  though  the 
facilities  for  aerial  infection  were  the  same.  The  disease  can 
also  be  produced  by  fleas  removed  from  plague  rats  and  trans- 
ferred directly  to  healthy  animals,  success  having  been  obtained 
in  fully  50  per  cent  of  experiments  of  this  kind.  When  plague- 
infected  guinea-pigs  are  placed  amongst  healthy  guinea-pigs 
comparatively  few  of  the  latter  acquire  the  disease  when  fleas 
are  absent  or  scanty ;  whereas  all  of  them  may  die  of  plague 
when  fleas  are  numerous.  This  result  demonstrates  the  com- 
paratively small  part  played  by  direct  contact,  even  when  of 
a  close  character.  Important  results  were  also  obtained  with 
regard  to  the  mode  of  infection  in  houses  where  there  had  been 
cases  of  plague.  It  was  found  possible  to  produce  the  disease 
in  susceptible  animals  by  means  of  fleas  taken  from  rats  in 

1  See  Journal  of  Hygiene,  \L  421  ;  vii.  323. 
28 


434  PLAGUE 

plague  houses.  When  animals  were  placed  in  plague  houses 
and  efficiently  protected  from  fleas  they  remained  healthy ; 
whereas  they  acquired  the  disease  when  the  cages  were  free 
to  the  access  of  fleas  in  the  neighbourhood. 

The  following  are  some  of  the  experiments  which  were  conducted.  A 
series  of  six  huts  were  built  which  only  differed  in  the  structure  of  their 
roofs.  In  two  the  roofs  were  made  of  ordinary  native  tiles  in  which  rats 
freely  lodge  ;  in  two  others  flat  tiles  were  used  in  which  rats  live,  but  in 
which  they  have  not  such  facilities  for  movement  as  in  the  first  set,  and  in 
the  third  pair  the  roof  was  formed  of  corrugated  iron.  Under  the  roof  in 
each  case  was  placed  a  wire  diaphragm  which  prevented  rats  or  their 
droppings  having  access  to  the  hut,  but  which  would  not  prevent  fleas 
falling  down  on  to  the  floor  of  the  hut.  The  huts  were  left  a  sufficient 
time  to  become  infected  with  rats,  and  then  on  the  floor  in  each  case 
healthy  guinea-pigs  mixed  with  guinea-pigs  artificially  infected  with 
plague  were  allowed  to  run  about  together.  In  the  first  two  sets  of  huts 
to  which  fleas  had  access  the  healthy  guinea-pigs  contracted  plague, 
while  in  the  third  set  they  remained  unaffected,  though  they  were  freely 
liable  to  contamination  by  contact  with  the  bodies  and  excreta  of  the 
diseased  animals.  In  the  third  set  of  huts  no  infection  took  place  as 
long  as  fleas  were  excluded,  but  when  accidentally  these  insects  obtained 
admission,  then  infection  of  the  uninoculated  animals  commenced. 
Other  experiments  were  also  performed.  In  one  case  healthy  guinea-pigs 
were  suspended  in  a  cage  two  inches  above  a  floor  on  which  infected  and 
flea-infested  animals  were  running  about.  Infection  occurred  in  the  cage, 
but  if  the  latter  were  suspended  at  a  distance  above  the  floor  higher  than 
a  flea  could  jump,  then  no  infection  took  place.  Again,  in  a  hut  in  which 
guinea-pigs  had  died  of  plague,  and  which  contained  infected  fleas,  two 
cages  were  placed,  each  containing  a  monkey.  One  cage  was  surrounded 
by  a  zone  of  sticky  material  broader  than  the  jump  of  a  flea.  The 
monkey  in  this  cage  remained  unaffected,  but  the  other  monkey  con- 
tracted plague. 

Other  experiments  showed  that  when  plague  bacilli  were 
placed  on  the  floors  of  houses,  they  died  off  in  a  comparatively 
short  period  of  time.  After  forty-eight  hours  it  was  not  found 
possible  to  reproduce  plague  by  inoculation  with  material  from 
floors  which  had  been  grossly  contaminated  with  cultures  of  the 
bacillus.  Afterwards,  however,  animals  placed  in  such  a  house 
might  become  infected  by  means  of  fleas.  In  all  these  experi- 
ments the  common  rat-flea  of  India — pulex  cheopis  (Rothschild) 
— was  used,  but  it  has  been  shown  that  this  flea,  when  a  rat  is 
not  available,  will  bite  a  man.  These  results  are  manifestly  of 
great  practical  importance.  They  show  that  direct  infection  by 
dust  and  other  material  through  small  lesions  of  the  skin  plays, 
probably,  a  comparatively  small  part  in  the  spread  of  the  disease, 
fleas  apparently  being  in  the  majority  of  cases  the  carriers  of  in- 
fection. They  also  point  to  important  preventive  measures, 
which  will  no  doubt  be  put  to  a  practical  test  before  long. 


TOXINS,    IMMUNITY,    ETC.  435 

In  primary  plague  pneumonia,  from  a  consideration  of  the 
anatomical  changes  and  the  clinical  facts,  the  disease  may  be 
said  to  be  produced  by  the  direct  passage  of  the  bacilli  into  the 
respiratory  passages.  Nevertheless  there  must  be  certain  factors, 
still  imperfectly  understood,  which  determine  the  incidence  of 
this  form ;  as  in  some  epidemics  of  the  highest  virulence  plague 
pneumonia  has  been  practically  absent,  though  opportunities  for 
infection  by  inhalation  must  have  been  present.  On  the  other 
hand,  a  case  of  plague  pneumonia  is  of  great  infectivity  in 
producing  other  cases  of  plague  pneumonia.  If  we  except 
infection  through  the  respiratory  passages  in  such  cases,  it  may 
be  said  that  direct  infection  from  patient  to  patient  is  relatively 
uncommon.  This  is  in  accordance  with  the  fact  that  in  bubonic 
plague  the  bacilli  are  not  discharged  from  the  unbroken  surface 
of  the  body,  and  are  only  present  in  the  secretions  in  severe  cases. 

The  occurrence  of  the  disease  in  rats  was  early  recognised,  and 
there  is  no  doubt  that  it  plays  a  very  important  part  in  the 
spread  of  epidemics.  The  disease  in  these  animals  has,  in  fact, 
been  the  means  of  rapidly  distributing  infection  over  wide  areas 
of  a  town  or  district.  This  has  been  abundantly  proved  in 
the  case  of  Bombay,  where  observations  have  shown  that  the 
migration  of  plague-infected  rats  to  quarters  comparatively  free 
from  the  disease,  has  been  followed  by  extensive  outbreaks  in 
these  places.  The  facts  stated  above  show  how  the  disease  is 
spread  among  these  animals  by  fleas,  and  how  it  is  conveyed 
by  them  to  the  human  subject. 

Toxins,  Immunity,  etc. — As  is  the  case  with  most  organisms 
which  extensively  invade  the  tissues,  the  toxins  in  plague  cultures 
are  chiefly  contained  in  the  bodies  of  the  bacteria.  Injection  of 
dead  cultures  in  animals  produces  distinctly  toxic  effects  ;  post 
mortem  hemorrhage  in  the  mucous  membrane  of  the  stomach, 
areas  of  necrosis  in  the  liver  and  at  the  site  of  inoculation,  may 
be  present.  The  toxic  substances  are  comparatively  resistant  to 
heat,  being  unaffected  by  an  exposure  to  65°  C.  for  an  hour.  By 
the  injection  of  dead  cultures  in  suitable  doses  a  certain  degree 
of  immunity  against  the  living  virulent  bacilli  is  obtained,  and, 
as  first  shown  by  Yersin,  Calmette,  and  Borrel,  the  serum  of 
such  immunised  animals  confers  a  degree  of  protection  on 
small  animals  such  as  mice.  On  these  facts  the  principles  of 
preventive  inoculation  and  serum  treatment,  presently  to  be 
described,  depend.  It  may  also  be  mentioned  that  the  filtrate 
of  a  plague  culture  possesses  a  very  slight  toxic  action,  and  the 
Indian  Plague  Commission  found  that  such  a  filtrate  has  practi- 
cally no  effect  in  the  direction  of  conferring  immunity. 


436  PLAGUE 

1.  Preventive  Inoculation — Hqfkine's  Method. — To  prepare 
the  preventive  fluid,  cultures  are  made  in  flasks  of  bouillon  with 
drops  of  oil  on  the  surface  (in  India  Haffkine  employed  a 
medium  prepared  by  digesting  goats'  flesh  with  hydrochloric 
acid  at  140°  C.  and  afterwards  neutralising  with  caustic  soda). 
In  such  cultures  stalactite  growths  (vide  supra)  form,  and  the 
flasks  are  shaken  every  few  days  so  as  to  break  up  the  stalactites 
and  induce  fresh  crops.  The  flasks  are  kept  at  a  temperature 
of  about  25°  C.,  and  growth  is  allowed  to  proceed  for  about 
six  weeks.  At  the  end  of  this  time  sterilisation  is  effected  by 
exposing  the  contents  of  the  flasks  to  65°  C.  for  an  hour; 
thereafter  carbolic  acid  is  added  in  the  proportion  of  *5  per  cent. 
The  contents  are  well  shaken  to  diffuse  thoroughly  the  sediment 
in  the  fluid,  and  are  then  distributed  in  small  sterilised  bottles  for 
use.  The  preventive  fluid  thus  contains  both  the  dead  bodies 
of  the  bacilli  and  any  toxins  which  may  be  in  solution.  It  is 
administered  by  subcutaneous  injection,  the  dose,  which  varies 
according  to  the  "strength,"  being  on  an  average  about  7 '5  c.c. 
Usually  only  one  injection  is  made,  sometimes  two ;  though  the 
latter  procedure  does  not  appear  to  have  any  advantage.  The 
method  has  been  systematically  tested  by  inoculating  a  certain 
proportion  of  the  inhabitants  of  districts  exposed  to  infection, 
leaving  others  uninoculated,  and  then  observing  the  proportion 
of  cases  of  disease  and  the  mortality  amongst  the  two  classes. 
The  results  of  inoculation,  as  attested  by  the  first  Indian 
Commission,  have  been  distinctly  satisfactory.  For  although 
absolute  protection  is  not  afforded  by  inoculation,  both  the 
proportion  of  cases  of  plague  and  the  percentage  mortality 
amongst  these  cases  have  been  considerably  smaller  in  the 
inoculated,  as  compared  with  the  uninoculated.  Protection 
is  not  established  till  some  days  after  inoculation,  and  lasts  for 
a  considerable  number  of  weeks,  possibly  for  several  months 
(Bannerman).  In  the  Punjab  during  the  season  1902-3  the  case 
incidence  among  the  inoculated  was  1*8  per  cent,  among  the 
uninoculated  7 '7  per  cent,  while  the  case  mortality  was  23 '9  and 
60 '1  per  cent  respectively  in  the  two  classes,  the  statistics  being 
taken  from  villages  where  10  per  cent  of  the  population  and 
upwards  had  been  inoculated. 

2.  Anti-plague  Sera. — Of  these,  two  have  been  used  as  therapeutic 
agents,  viz.  that  of  Yersin  and  that  of  Lustig.  Yersin's  serum  is 
prepared  by  injections  of  increasing  doses  of  plague  bacilli  into  the 
horse.  In  the  early  stages  of  immunisation  dead  bacilli  are  injected 
subcutaneously,  thereafter  into  the  veins,  and,  finally,  living  bacilli  are 
injected  intravenously.  After  a  suitable  time  blood  is  drawn  off  and 
the  serum  is  preserved  in  the  usual  way.  Of  this  serum  10-20  c.c. 


METHODS   OF   DIAGNOSIS  437 

are  used,  and  injections  are  usually  repeated  on  subsequent  days. 
Lustig's  serum  is  prepared  by  injecting  a  horse  with  repeated  and 
increasing  doses  of  a  substance  derived  from  the  bodies  of  plague  bacilli, 
probably  in  great  part  nucleo-proteid.  Masses  of  growth  are  obtained 
from  the  surface  of  agar  cultures,  and  are  broken  up  and  dissolved  in  a 
1  per  cent  solution  of  caustic  potash.  The  solution  is  then  made  slightly 
acid  by  hydrochloric  acid,  when  a  bulky  precipitate  forms  ;  this  is 
collected  on  a  filter  and  dried.  For  use  a  weighed  amount  is  dissolved 
in  a  weak  solution  of  carbonate  of  soda  and  then  injected.  The  serum 
is  obtained  from  the  animal  in  the  usual  way.  Extensive  observations 
with  both  of  these  sera  show  that  neither  of  them  can  be  considered 
a  powerful  remedy  in  cases  of  plague,  though  in  certain  instances 
distinctly  favourable  results  have  been  recorded.  The  Indian  Com- 
mission, however,  came  to  the  conclusion  "  that,  on  the  whole,  a  certain 
amount  of  advantage  accrued  to  the  patients  both  in  case  of  those 
injected  with  Yersin's  serum  and  of  those  injected  with  Lustig's  serum." 
It  may  also  be  mentioned  that  the  Commission  found,  as  the  result  of 
experiments,  that  Yersin's  serum  modified  favourably  the  course  of  the 
disease  in  animals,  whereas  Lustig's  serum  had  no  such  effect 

3.  Serum  Diagnosis, — Specific  agglutinins  may  appear  in  the  blood  of 
patients  suffering  from  plague,  as  also  they  do  in  the  case  of  animals 
immunised  against  the  plague  bacillus.  It  is  to  be  noted,  however,  that 
in  clinical  cases  the  reaction  is  not  invariably  present,  the  potency  of 
the  serum  is  not  of  high  order,  and  the  carrying  out  of  the  test  is  com- 
plicated by  the  natural  tendency  of  the  bacilli  to  cohere  in  clumps.  For 
the  last  reason  the  macroscopic  (sedimentation)  method  is  be  preferred 
to  the  microscopic  (p.  111).  A  suspension  of  plague  bacilli  is  made  by 
breaking  up  a  young  agar  culture  in  75  per  cent  sodium  chloride 
solution  ;  the  larger  Hocculi  of  growth  are  allowed  to  settle,  and  the 
fine,  supernatant  emulsion  is  employed  in  the  usual  way.  According  to 
the  results  of  the  German  Plague  Commission  and  the  observations  of 
Cairns,  made  during  the  Glasgow  epidemic,  it  may  be  said  that  the 
reaction  is  best  obtained  with  dilutions  of  the  serum  of  from  1  : 10  to 
1  : 50.  Cairns  found  that  the  date  of  its  appearance  is  about  a  week 
after  the  onset  of  illness,  and  that  it  usually  increases  till  about  the  end 
of  the  sixth  week,  thereafter  fading  off.  It  is  most  marked  in  severe 
cases  characterised  by  an  early  and  favourable  crisis,  less  marked  in 
severe  cases  ultimately  proving  fatal,  whilst  in  very  mild  cases  it  is 
feeble  or  may  be  absent.  The  method,  if  carefully  applied,  may  be  of 
service  under  certain  conditions  ;  but  it  will  be  seen  that  its  use  as  a 
means  of  diagnosis  is  somewhat  restricted. 

Methods  of  Diagnosis. — Where  a  bubo  is  present  a  little  of 
the  juice  may  be  obtained  by  plunging  a  sterile  hypodermic 
needle  into  the  swelling.  The  fluid  is  then  to  be  examined  micro- 
scopically, and  cultures  on  agar  or  blood  serum  should  be  made 
by  the  successive  stroke  method.  The  cultural  and  morpho- 
logical characters  are  then  to  be  investigated,  the  most  important 
being  the  involution  forms  on  salt  agar  and  the  stalactite 
growth  in  bouillon,  though  the  latter  may  not  always  be  obtained 
with  the  plague  bacillus  :  the  pathogenic  properties  should  also 
be  studied,  the  guinea-pig  being  on  the  whole  most  suitable  for 


438  RELAPSING  FEVER  AND  AFRICAN  TICK  FEVER 

subcutaneous  inoculation.  In  many  cases  a  diagnosis  may  be 
made  by  microscopic  examination  alone,  as  in  no  other  known 
condition  than  plague  do  bacilli  with  the  morphological  char- 
acters of  the  plague  bacillus  occur  in  large  numbers  in  the  lym- 
phatic glands.  The  organism  may  be  obtained  in  culture  from 
the  blood  in  a  considerable  proportion  of  cases  by  withdrawing 
a  few  cubic  centimetres  and  proceeding  in  the  usual  manner. 
On  the  occurrence  of  the  first  suspected  case,  every  care  to 
exclude  possibility  of  doubt  should  be  used  before  a  positive 
opinion  is  given. 

In  a  case  of  suspected  plague  pneumonia,  in  addition  to 
microscopic  examination  of  the  sputum,  the  above  cultural 
methods  along  with  animal  inoculation  with  the  sputum  should 
be  carried  out ;  subcutaneous  injection  in  the  guinea-pig  and 
smearing  the  nasal  mucous  membrane  of  the  rat  may  be  recom- 
mended. Here  a  positive  diagnosis  should  not  be  attempted  by 
microscopic  examination  alone,  especially  in  a  plague-free  dis- 
trict, as  bacilli  morphologically  resembling  the  plague  organism 
may  occur  in  the  sputum  in  other  conditions. 

RELAPSING  FEVER  AND  AFRICAN  TICK  FEVER. 

At  a  comparatively  early  date,  namely  in  1873,  when  practi- 
cally nothing  was  known  with  regard  to  the  production  of  disease 
by  bacteria,  a  highly  characteristic  organism  was  discovered  in 
the  blood  of  patients  suffering  from  relapsing  fever.  This 
discovery  was  made  by  Obermeier,  and  the  organism  is  usually 
known  as  the  spirillum  or  spirochoete  Obermeieri,  or  the  spirillum 
of  relapsing  fever.  He  described  its  microscopical  characters, 
and  found  that  its  presence  in  the  blood  had  a  definite  relation 
to  the  time  of  the  fever,  as  the  organism  rapidly  disappeared 
about  the  time  of  the  crisis,  and  reappeared  when  a  relapse 
occurred.  He  failed  to  find  such  an  organism  in  any  other 
disease.  His  observations  were  fully  confirmed,  and  his  views 
as  to  its  causal  relationship  to  the  disease  were  generally  accepted. 
Later,  the  disease  was  produced  in  the  human  subject  by  inocula- 
tions with  blood  containing  the  organisms,  and  a  similar  con- 
dition has  been  produced  in  apes. 

Recently  it  has  been  shown  that  the  so-called  "tick  fever" 
prevalent  in  Africa  is  also  due  to  a  spirochaete  of  similar 
character,  and  results  of  the  highest  importance  have  been 
established  with  regard  to  the  part  played  by  ticks  in  the  trans- 
mission of  the  disease.  Doubt  still  obtains  as  to  the  relationship 
of  the  organisms  of  the  two  diseases,  but  all  are  agreed  that  they 


CHARACTERS   OF   THE   SPIRILLUM  439 

are  closely  similar,  if  not  identical.  As  a  matter  of  convenience, 
and  in  accordance  with  the  history  of  the  investigations,  we  shall 
give  the  chief  facts  regarding  these  diseases  separately.  It  has 
also  been  shown  that  spirillar  diseases  or  "  spirilloses,"  as  they 
are  called,  are  widespread  in  the  animal  kingdom.  Such 
infections  have  been  described  in  geese  by  SacharofF,  in  fowls  by 
Marchoux  and  Salimbeni,  in  oxen  and  sheep  by  Theiler,  and  in 
bats  by  Nicolle  and  Comte,  and  it  is  interesting  to  note  that  in 
the  case  of  the  spirilloses  of  oxen  and  fowls  the  infection  is 
transmissible  by  means  of  ticks. 

The  work  of  Schaudinn(v.  p.  548)  has  led  to  these  spirochsete 
being  regarded  by  various  authorities  as  members  of  the  protozoal 
group.  As,  however, 
longitudinal  division  has 
not  been  satisfactorily 
observed  and  no  cycle  of 
development  has  been 
determined  amongst  them, 
we  are  not  justified  at 
present  in  removing  them 
from  the  class  of  bacteria. 

Characters     of     the 
Spirillum.  —  The    organ- 
isms as  seen  in  the  blood 
during  the  fever  are  deli- 
cate spiral  filaments  which 
have  a  length  of  from  two 
to  six  times  the  diameter 
of  a  red  blood  corpuscle.    Fia    149.— Spirilla    of    relapsing    fever    in 
They  are,  however,  exceed-         human  blood.    Film  preparation.    (After 
ingly  thin,  their  thickness        Koch.)     x  about  1000. 
being  much  less  than  that 

of  the  cholera  spirillum.  They  show  several  regular  sharp  curves 
or  windings,  of  number  varying  according  to  the  length  of  the 
spirilla,  and  their  extremities  are  finely  pointed  (Fig.  149). 
There  are  often  to  be  seen  in  the  spirals,  portions  which  are 
thinner  and  less  deeply  stained  than  the  rest,  and  which  suggest 
the  occurrence  of  transverse  division.  They  are  actively  motile, 
and  may  be  seen  moving  quickly  across  the  microscopic  field 
with  a  peculiar  movement  which  is  partly  twisting  and  partly 
undulatory,  and  disturbing  the  blood  corpuscles  in  their  course. 
Novy  and  Knapp  have  found  that  there  is  a  single  flagellum  at 
one  end  of  the  organism. 

They  stain  with  watery  solutions  of  the  basic  aniline  dyes, 


440  RELAPSING  FEVER 

though  somewhat  faintly,  and  are  best  coloured  by  the 
Romanowsky  method  or  one  of  its  modifications.  When  thus 
stained  they  usually  have  a  uniform  appearance  throughout,  or 
may  be  slightly  granular  at  places,  but  they  show  no  division 
into  short  segments.  They  lose  the  stain  in  Gram's  method. 

In  blood  outside  the  body  the  organisms  have  a  considerable 
degree  of  vitality,  and  when  kept  in  sealed  tubes  they  have  been 
found  alive  and  active  after  many  days.  They  are  readily 
killed  at  a  temperature  of  60°  C.,  but  may  be  exposed  to  0°  C. 
without  being  killed.  There  is  no  evidence  that  they  form  spores. 

Relations  to  the  Disease. — In  relapsing  fever,  after  a  period 
of  incubation  there  occurs  a  rapid  rise  of  temperature  which 
lasts  for  about  five  to  seven  days.  At  the  end  of  this  time  a 
crisis  occurs,  the  temperature  falling  quickly  to  normal.  In  the 
course  of  about  other  seven  days  a  sharp  rise  of  temperature 
again  takes  place,  but  on  this  occasion  the  fever  lasts  a  shorter 
time,  again  suddenly  disappearing.  A  second  or  even  third 
relapse  may  occur  after  a  similar  interval.  The  spirilla  begin  to 
appear  in  the  blood  shortly  before  the  onset  of  the  pyrexia,  aud 
during  the  rise  of  temperature  rapidly  increase  in  number. 
They  are  very  numerous  during  the  fever,  a  large  number  being 
often  present  in  every  field  of  the  microscope  when  the  blood  is 
examined  at  this  stage.  They  begin  to  disappear  shortly  before 
the  crisis :  after  the  crisis  they  are  entirely  absent  from  the 
circulating  blood.  A  similar  relation  between  the  presence  of 
the  spirilla  in  the  blood  and  the  fever  is  found  in  the  case  of  the 
relapses,  whilst  between  these  they  are  entirely  absent.  Munch 
in  1876  produced  the  disease  in  the  human  subject  by  injecting 
blood  containing  the  spirilla,  and  this  experiment  has  been 
several  times  repeated  with  the  same  result.  Additional  proof, 
that  the  organism  is  the  cause  of  the  disease  has  been  afforded  by 
experiments  on  animals.  Carter  in  1879  was  the  first  to  show  that 
the  disease  could  be  readily  produced  in  monkeys,  and  his  experi- 
ments were  confirmed  by  Koch.  In  such  experiments  the  blood 
taken  from  patients  and  containing  the  spirilla  was  injected  sub- 
cutaneously.  In  the  disease  thus  produced  there  is  an  incubation 
period  which  usually  lasts  about  three  days.  At  the  end  of  that 
time  the  spirilla  rapidly  appear  in  the  blood,  and  shortly  after- 
wards the  temperature  quickly  rises.  The  period  of  pyrexia 
usually  lasts  for  two  or  three  days,  and  is  followed  by  a  marked 
crisis.  As  a  rule  there  is  no  relapse,  but  occasionally  one  of 
short  duration  occurs.1  White  mice  and  rats  are  also  susceptible 

1  Norris,  Pappenheimer,  and  Flournoy,  in  their  experiments  on  monkeys 
in  America,  found  that  several  relapses  occurred. 


IMMUNITY  441 

to  infection.     In  the  former  animals  the  disease  is  characterised 
by  several  relapses,  in  the  latter  there  is,  however,  no  relapse. 

Numerous  attempts  to  cultivate  this  organism  outside  the 
body  have  all  been  attended  with  failure,  and  it  has  been 
abundantly  shown  that  it  does  not  grow  on  any  of  the  media 
ordinarily  in  use.  Koch  found  that  on  blood  serum  the 
filaments  of  the  spirilla  increased  somewhat  in  length,  and 
formed  a  sort  of  felted  mass,  but  that  no  multiplication  took 


" 


FIG.  150. — Spirillum  Obermeieri  in  blood  of  infected  mouse.      x  1000. 

place.  Recently  Norris,  Pappenheimer,  and  Flournoy  have 
found  that  a  considerable  amount  of  multiplication  may  take 
place  in  the  citrated  blood  of  man  and  the  rat. 

Immunity. — Metchnikoff  found  that  during  the  fever  the 
spirilla  were  practically  never  taken  up  by  the  leucocytes  in  the 
circulating  blood,  but  that  at  the  time  of  the  crisis,  on  dis- 
appearing from  the  blood,  they  accumulated  in  the  spleen  and 
were  ingested  in  large  numbers  by  the  microphages  or  poly- 
morphonuclear  leucocytes.  Within  these  they  rapidly  under- 
went degeneration  and  disappeared.  It  is  to  be  noted  in  this 
connection  that  swelling  of  the  spleen  is  a  very  marked  feature 
in  relapsing  fever.  These  observations  were  entirely  confirmed 


442  RELAPSING  FEVER 

by  Soudake witch,  who  also  produced  the  disease  in  two  monkeys 
(cercocebus  fuliginosus)  from  which  the  spleen  had  been  previously 
removed,  the  animals  having  been  allowed  to  recover  completely 
from  the  operation,  and  found  that  in  these  cases  the  spirilla 
did  not  disappear  from  the  blood  at  the  usual  time,  but  rather 
increased  in  number,  and  a  fatal  result  followed  on  the  eighth 
and  ninth  days  respectively.  Recent  observations,  however,  in- 
dicate that,  as  in  the  case  of  so  many  other  diseases,  the  all- 
important  factor  in  the  destruction  of  the  organisms  is  the 
development  of  antagonistic  substances  in  the  blood.  Lamb 
found  in  the  case  of  the  monkey  (macacus  radiatus)  that  the 
removal  of  the  spleen  of  an  animal  rendered  immune  by  an 
attack  of  the  disease  did  not  render  it  susceptible  to  fresh 
inoculation,  and  attributed  the  immunity  to  the  presence  of 
bactericidal  bodies  in  the  serum.  He  found,  for  example,  that 
in  vitro  the  serum  of  an  immune  animal  brought  the  movements 
of  the  spirilla  to  an  end,  clumped  them,  and  caused  their  dis- 
integration ;  and  further,  that  when  the  spirilla  and  the  immune 
serum  were  injected  in  one  case  into  a  fresh  monkey  no  disease 
developed.  In  opposition  to  Soudake  witch,  Lamb  found  that 
with  a  monkey  from  which  the  spleen  had  been  removed  death 
did  not  occur  after  it  was  inoculated  with  the  spirilla.  Sawt- 
schenko  and  Milkich  found  that  there  are  developed  during  the 
disease  an  immune  body  and  an  agglutinin,  while  Novy  and 
Knapp  in  their  recent  important  work  distinguish  germicidal, 
immunising,  and  agglutinating  substances.  They  found  that 
the  blood  of  the  rat  has  no  germicidal  properties  during  the 
onset  of  the  disease,  but  that  these  appear  and  become  well 
marked  during  the  decline.  They  produced  a  "  hyper-immunity  " 
in  rats  by  repeated  injections  of  blood  containing  the  spirilla, 
and  found  that  the  serum  of  such  animals  had  a  markedly  cura- 
tive effect,  and  could  cut  short  the  disease  in  rats,  mice,  and 
monkeys. 

In  the  case  of  the  human  subject  it  has  been  found  that  a 
second  attack  of  the  disease  can  follow  the  first  after  a  com- 
paratively short  period  of  time,  and  it  is  often  said  that  one 
attack  does  not  confer  immunity.  It  is  probably  rather  the  case 
that  the  immunity  conferred  is  of  very  short  duration.  The 
course  of  events  in  the  disease  might  be  explained  by  supposing 
that  immunity  of  short  duration  is  produced  during  the  first 
period  of  pyrexia,  but  that  it  does  not  last  until  all  the  spirilla 
have  been  destroyed,  some  still  surviving  in  internal  organs 
or  in  tissues  where  they  escape  the  bactericidal  action  of  the 
serum.  With  the  disappearance  of  the  immunity  the  organ- 


AFRICAN   TICK   FEVER  443 

isms  reappear  in  the  blood,  the  relapse  being,  however,  of 
shorter  duration  and  less  severe  than  the  first  attack.  This  is 
repeated  till  the  immunity  lasts  long  enough  to  allow  all  the 
organisms  to  be  killed.  The  production  of  anti-substances 
during  the  febrile  attack  is  an  established  fact,  and  the  experi- 
mental results  above  detailed  show  that  the  disease  as  met  with 
in  the  human  subject  will  probably  be  eminently  amenable  to 
serum  therapeutics. 

The  fact  that  other  like  spirillar  diseases  may  be  conveyed 
by  the  bites  of  insects  makes  it  extremely  probable  that  relaps- 
ing fever  may  also  be  transmitted  in  this  way,  and  a  number  of 
facts  point  to  the  bed-bug  as  the  means  of  transmission.  The 
presence  of  the  spirilla  within  the  bodies  of  bugs  has  been 
demonstrated,  and  it  has  also  been  shown  that  they  may  be 
present  for  a  considerable  time  after  the  insects  have  sucked  the 
blood, — according  to  Karlinski  for  forty  days.  Tictin,  by  inject- 
ing the  blood  removed  from  a  number  of  bugs  which  had  been 
allowed  to  bite  infected  monkeys,  produced  the  disease  in  other 
healthy  monkeys,  but  so  far  as  we  know  the  crucial  experiment 
of  infecting  man  by  means  of  the  bites  of  these  insects  has  not 
yet  been  successfully  carried  out. 

African  Tick  Fever. 

The  disease  long  known  by  this  name  as  prevalent  in  Africa 
has  also  been  shown  to  be  caused  by  a  spirillum  or  spirochaete. 
Organisms  of  this  nature  had  been  seen  in  the  blood  of  patients 
in  Uganda  by  Greig  and  Nabarro  in  1903,  and  Milne  and  Ross 
in  the  end  of  1904  recorded  a  series  of  observations  which  led 
them  to  the  conclusion  that  tick  fever  was  due  to  a  spirochsete. 
It  is,  however,  chiefly  owing  to  the  work  of  Button  and  Todd  in 
the  Congo  Free  State,  on  the  one  hand,  and  of  Koch  in  German 
East  Africa,  on  the  other,  that  our  knowledge  of  this  disease  has 
been  thoroughly  established.  The  former  gave  a  full  account  of 
the  organism,  and  by  means  of  experiments  showed  that  the 
disease  could  be  transferred  by  means  of  ticks  to  healthy 
animals.  The  latter  published  interesting  observations  on  the 
infection  of  the  ticks  and  the  transmission  of  the  organisms  to 
the  young,  and  also  important  facts  with  regard  to  the  extent  to 
which  ticks  were  infected  in  certain  districts. 

The  following  are  the  chief  facts  regarding  this  fever. 
Clinically  the  fever  closely  resembles  relapsing  fever,  but  the 
periods  of  fever  are  somewhat  shorter,  rarely  lasting  for  more 
than  two  or  three  days.  It  is  rarely  attended  with  a  fatal  result 


444 


AFRICAN  TICK  FEVER 


unless  in  patients  debilitated  by  other  causes.  The  spirilla  are 
considerably  fewer  in  the  blood  than  in  the  European  relapsing 
fever,  and  sometimes  a  careful  search  may  be  necessary  before  they 
are  found.  Morphologically  they  are  said  to  be  practically 
identical,  although  Koch  thought  that  the  organisms  in  tick 
fever  tended  on  the  whole  to  be  slightly  longer ;  the  average 
length  may  be  said  to  be  15  to  35  /x.  Button  and  Todd  showed 
that  it  was  possible  to  transmit  the  disease  to  certain  monkeys 


FIG.  151. — Film  of  human  blood  containing  spirillum  of  tick  fever,     x  1000.1 

(cercopitheci)  by  means  of  ticks  which  had  been  allowed  to  bite 
patients  suffering  from  the  disease,  the  symptoms  in  these 
animals  appearing  about  five  days  after  inoculation.  The  disease 
thus  produced  is  characterised  by  several  relapses,  and  often 
leads  to  a  fatal  result.  In  one  case  they  produced  the  disease 
by  means  of  young  ticks  hatched  from  the  eggs  of  ticks  which 
had  been  allowed  to  suck  the  blood  of  fever  patients,  and  they 
came  to  the  conclusion  that  the  spirilla  were  not  simply  carried 
mechanically  by  the  ticks,  but  probably  underwent  some  cycle  of 

1  We  are  indebted  to  Colonel  Leishman,  R.A.  M.C.,  for  the  preparations 
from  which  Figs.  150-152  were  taken. 


AFRICAN   TICK  FEVER  445 

development  in  the  tissues  of  the  latter.  The  species  of  tick 
concerned  is  the  ornithodorus  moubata.  These  results  were  con- 
firmed and  extended  by  Koch.  He  found  that  after  the  ticks 
had  been  allowed  to  suck  the  blood  containing  the  organisms, 
these  could  be  found  for  a  day  or  two  in  the  stomachs  of  the 
insects.  After  this  time  they  gradually  disappeared  from  the 
stomach,  but  were  detected  in  large  numbers  in  the  ovaries  of 
the  female  ticks,  where  they  sometimes  formed  felted  masses. 


FIG.  152. — Spirillum  of  human  tick  fever  (Spirillum  Duttoni)  in  blood  of 
infected  mouse,      x  1000. 

He  also  traced  the  presence  of  the  spirilla  in  the  eggs  laid  by  the 
infected  ticks,  and  in  the  young  embryos  hatched  from  them. 
He  was  thus  able  to  demonstrate  how  the  infection  might  be 
continued  within  the  tissues  of  ticks  from  generation  to 
generation ;  in  the  process  of  transmission,  however,  the  spirillar 
form  was  always  observed,  and  there  was  no  evidence  that  the 
organism  went  through  a  cycle  of  change.  Koch  also  made 
extensive  observations  on  the  ticks  in  German  East  Africa,  and 
found  that  of  over  six  hundred  examined  11  per  cent  of  these 
insects  along  the  main  caravan  routes  contained  spirilla,  and 
in  some  localities  almost  half  of  the  ticks  were  infected.  In 


446  MALTA  FEVER 

places  removed  from  the  main  lines  of  commerce  he  still  found 
them,  though  in  smaller  number.  It  has  also  been  demonstrated 
that  in  some  places  the  ticks  are  found  to  be  infected  with  the 
spirilla  although  the  inhabitants  do  not  suffer  from  tick  fever,  a 
circumstance  which  is  probably  due  to  an  acquired  immunity 
against  the  disease. 

Although  our  knowledge  regarding  the  relationships  of  these 
to  other  spirilla  is  far  from  complete,  certain  differences  between 
the  organisms  of  European  relapsing  fever  and  of  African  tick 
fever  have  been  established.  Zettnow,  for  example,  has  found 
that  the  organism  of  tick  fever  possesses  numerous  lateral 
flagella,  whereas,  as  already  stated,  the  sp.  Obermeieri  has  a 
single  terminal  flagellum.  This  observation,  however,  has  not 
yet  been  confirmed.  Differences  are  also  brought  out  by  animal 
inoculation.  In  addition  to  the  more  severe  illness  produced  by 
the  spirillum  of  tick  fever  in  monkeys,  it  has  been  found  by 
Breinl  and  Kinghorn  that  a  considerable  number  of  animals 
are  susceptible  to  the  African  spirillum,  including  rabbits  and 
guinea-pigs,  which  appear  to  be  refractory  to  the  organism  of 
relapsing  fever.  Breinl  also  compared  the  immunity  conferred 
by  the  sp.  Obermeieri  and  by  the  tick  fever  spirillum,  and  found 
that  each  conferred  a  relative  active  immunity  against  itself,  but 
not  against  the  other.  It  is  thus  highly  probable  that  they 
represent  two  distinct  species.  Spirillar  fever  has  also  been 
found  in  India,  but  its  relations  to  the  European  and  African 
fevers  have  not  yet  been  fully  worked  out. 


MALTA  FEVER. 

Synonyms — Mediterranean  Fever  :  Rock  Fever  of  Gibraltar  : 
Neapolitan  Fever,  etc. 

This  disease  is  of  common  occurrence  along  the  shores  of  the 
Mediterranean  and  in  its  islands.  Since  its  bacteriology  has 
been  worked  out,  it  has  been  found  to  occur  also  in  India,  China, 
and  in  some  parts  of  North  and  South  America,  its  distribution 
being  much  wider  than  was  formerly  supposed.  Although  from 
its  symptomatology  and  pathological  anatomy  it  had  been  re- 
cognised as  a  distinct  affection,  and  was  known  under  various 
names,  its  precise  etiology  was  unknown  till  the  publication 
of  the  researches  of  Colonel  Bruce  in  1887.  From  the  spleen 
of  patients  dead  of  the  disease  he  cultivated  a  characteristic 
organism,  now  known  as  the  micrococcus  melitensis,  and  by 


MICROCOCCUS   MELITENSIS  447 

means  of  inoculation  experiments  established  its  causal  relation- 
ship to  the  disease.  Wright  and  Semple  applied  the  agglutina- 
tion test  to  the  diagnosis  of  the  disease,  while  within  recent 
years  the  mode  of  spread  of  the  disease  has  been  fully  studied 
by  a  Commission,  and  it  has  been  demonstrated  that  goat's  milk 
is  the  chief  means  of  infection. 

The  duration  of  the  disease  is  usually  long — often  two  or 
three  months,  though  shorter  and  much  longer  periods  are  met 
with.  Its  course  is  very  variable,  the  fever  being  of  the  con- 
tinued type  with  irregular  remissions.  In  addition  to  the  usual 
symptoms  of  pyrexia,  there  occur  profuse  perspirations,  pains 
and  sometimes  swellings  in  the  joints,  occasionally  orchitis, 
whilst  constipation  is  usually  a  marked  feature.  The  mortality 
is  low — about  2  per  cent  (Bruce). 

In  fatal  cases  the  most  striking  post-mortem  change  is  in  the 
spleen.  This  organ  is  enlarged,  often  weighing  slightly  over  a 
pound,  and  in  a  condition  of  acute  congestion ;  the  pulp  is  soft 
and  may  be  diffluent,  and  the  Malpighian  bodies  are  swollen  and 
indistinct.  In  the  other  organs  the  chief  change  is  cloudy 
swelling ;  in  the  kidneys  there  may  be  in  addition  glomerular 
nephritis.  The  lymphoid  tissue  of  the  intestines  shows  none  of 
the  changes  characteristic  of  typhoid  fever. 

Micrococcus  Melitensis. — This  is  a  small,  rounded,  or  slightly 
oval  organism  about  '4  /A  in  diameter,  which  is  specially  abundant 
in  the  spleen.  It  usually  occurs  singly  or  in  pairs,  but  in 
cultures  short  chains  are  also  met  with  (Fig.  153).  (Durham 
has  shown  that  in  old  cultures  kept  at  the  room  temperature 
bacillary  forms  appear,  and  we  have  noticed  indications  of  such 
in  comparatively  young  cultures ;  the  usual  form  is,  however, 
that  of  a  coccus.)  It  stains  fairly  readily  with  the  ordinary 
basic  aniline  stains,  but  loses  the  stain  in  Gram's  method.  It  is 
generally  said  to  be  a  non-motile  organism.  Gordon,  however, 
is  of  a  contrary  opinion,  and  has  recently  demonstrated  that  it 
possesses  from  one  to  four  flagella,  which,  however,  are  difficult 
to  stain.  In  the  spleen  of  a  patient  dead  of  the  disease  it  occurs 
irregularly  scattered  through  the  congested  pulp ;  it  may  also  be 
found  in  small  numbers  post  mortem  in  the  capillaries  of  various 
organs.  It  may  be  cultivated  from  the  blood  during  life  in  a 
considerable  proportion  of  cases;  for  this  purpose  5-10  c.c.  of 
blood  should  be  withdrawn  from  a  vein  and  distributed  in  small 
flasks  of  bouillon.  The  micrococcus  was  found  by  the  members 
of  the  Commission  in  the  urine  of  Malta  fever  patients  in  10  per 
cent  of  the  cases  examined ;  it  was  sometimes  scanty,  but  some- 
times present  in  large  numbers. 


448 


MALTA   FEVER 


Cultivation. — This  can  usually  readily  be  effected  by  making 
stroke  cultures  on  agar  tubes  from  the  spleen  pulp  and  incub- 
ating at  37°  C.  The  colonies,  which  are  usually  not  visible 
before  the  third  or  fourth  day,  appear  as  small  round  discs, 
slightly  raised  and  of  somewhat  transparent  appearance.  The 
maximum  size — 2-3  mm.  in  diameter — is  reached  about  the 
ninth  day ;  at  this  period  by  reflected  light  they  appear  pearly 
white,  while  by  transmitted  light  they  have  a  yellowish  tint  in 
the  centre,  bluish  white  at  the  periphery.  A  stroke  culture 
shows  a  layer  of  growth  of  similar  appearance  with  somewhat 

serrated  margins.  Old 
cultures  assume  a  buff  tint. 
The  optimum  temperature 
is  37°  C.,  but  growth  still 
occurs  down  to  about  20° 
C.  On  gelatin  at  summer 
temperature  growth  is  ex- 
tremely slow — after  two  or 
three  weeks,  in  a  puncture 
culture,  there  is  a  delicate 
line  of  growth  along  the 
needle  track  and  a  small 
flat  expansion  of  growth 
on  the  surface.  There  is 
no  liquefaction  of  the 
medium.  In  bouillon  there 
occurs  a  general  turbidity 
with  flocculent  deposit  at 
the  bottom ;  on  the  surface 

there  is  no  formation  of  a  pellicle.  The  reaction  of  the  media 
ought  to  be  very  faintly  alkaline,  as  marked  alkalinity  interferes 
with  the  growth.  On  potatoes  no  visible  growth  takes  place 
even  at  the  body  temperature,  though  the  organism  multiplies 
to  a  certain  extent.  Outside  the  body  the  organism  has 
considerable  powers  of  vitality,  as  it  has  been  found  to  sur- 
vive in  a  dry  condition  in  dust  and  clothing  for  a  period  of  two 
months. 

Eelations  to  the  Disease. — There  is  in  the  first  place  ample 
evidence,  from  examination  of  the  spleen,  both  post  mortem  and 
during  life,  that  this  organism  is  always  present  in  the  disease. 
The  experiments  of  Bruce  and  Hughes  first  showed  that  by 
inoculation  with  even  comparatively  small  doses  of  pure  cultures 
the  disease  could  be  produced  in  monkeys,  sometimes  with  a 
fatal  result.  And  it  has  now  been  fully  established  that  inocula- 


FIG.  153. — Micrococcus  raelitensis,  from  a 

two  days'  culture  on  agar  at  37°  C. 

Stained  with  fuchsin.      x  1000. 


MODE   OF   SPREAD   OF   THE   DISEASE         449 

tion  with  the  minutest  amount  of  culture,  even  by  scarification, 
leads  to  infection  both  in  monkeys  and  in  the  human  subject. 

Rabbits,  guinea-pigs,  and  mice  are  insusceptible  to  inocula- 
tion by  the  ordinary  method.  Durham,  by  using  the  intra- 
cerebral  method  of  inoculation,  has,  however,  succeeded  in 
raising  the  virulence  so  that  the  organism  is  capable  of  produc- 
ing in  guinea-pigs  on  intra-peritoneal  injection  illness  with  some- 
times a  fatal  result  many  weeks  afterwards.  An  interesting 
point  brought  out  by  these  experiments  is  that,  in  the  case  of 
animals  which  survive,  the  micrococcus  may  be  cultivated  from 
the  urine  several  months  after  inoculation. 

Mode  of  Spread  of  the  Disease. — The  work  of  the  recent 
Commission  has  resulted  in  establishing  facts  of  the  highest 
importance  with  regard  to  the  spread  of  the  disease.  In  the 
course  of  investigations  Zammitt  found  that  the  blood  of  many 
of  the  goats  agglutinated  the  micrococcus  melitensis,  and 
Horrocks  obtained  cultures  of  the  organism  from  the  milk. 
Further  observations  showed  that  agglutination  was  given  in 
the  case  of  50  per  cent  of  the  goats  in  Malta,  whilst  the  organism 
was  present  in  the  milk  in  10  per  cent.  Sometimes  the  organism 
was  present  in  enormous  numbers,  and  in  these  cases  the  animal 
usually  appeared  poorly  nourished,  whilst  the  milk  had  a  some- 
what serous  character.  In  other  cases,  however,  the  organism 
was  found  when  the  animals  appeared  healthy  and  there  was 
no  physical  or  chemical  change  in  the  milk.  It  was  also 
determined  that  the  organism  might  be  excreted  for  a  period 
of  two  to  three  months  before  any  notable  change  occurred  in 
the  milk.  Agglutination  is  usually  given  by  the  milk  of  infected 
animals,  and  this  property  was  always  present  when  the  micro- 
coccus  was  found  in  the  milk.  It  was,  moreover,  found  that  mon- 
keys and  goats  could  be  readily  infected  by  feeding  them  with 
milk  containing  the  micrococcus,  the  disease  being  contracted 
by  fully  80  per  .cent  of  the  monkeys  used.  It  was  therefore 
rendered  practically  certain  that  the  human  subject  was  infected 
by  means  of  such  milk,  and  the  result  of  preventive  measures 
by  which  milk  was  excluded  as  an  article  of  dietary  amongst 
the  troops  in  Malta  has  fully  borne  out  this  view.  After  such 
measures  were  instituted,  the  number  of  cases  in  the  second 
half  of  1906  fell  to  11  per  thousand,  as  contrasted  with  47 
per  thousand  in  the  corresponding  part  of  the  preceding  year. 
The  various  facts  with  regard  to  the  epidemiology  of  the  disease 
have  thus  been  cleared  up.  For  example,  it  is  more  prevalent 
in  the  summer  months,  when  more  milk  is  consumed,  and  there 
is  a  larger  proportion  of  cases  amongst  those  in  good  social 
29 


450  MALTA   FEVER 

position,  the  officers,  for  example,  suffering  more  in  proportion 
than  the  privates.  Another  interesting  fact,  pointed  out  by 
Horrocks,  is  that  the  disease  has  practically  disappeared  from 
Gibraltar  since  the  practice  of  importing  goats  from  Malta  has 
stopped. 

The  work  of  the  Commission,  so  far  as  it  has  gone,  has 
been  to  exclude  other  modes  of  infection  as  being  of  practical 
importance,  by  dust,  by  the  bites  of  mosquitoes,  etc.,  and  if  it 
is  conveyed  by  contact  at  all,  this  is  only  when  the  contact  is 
of  an  intimate  character,  and  even  then  it  is  probably  of  rare 
occurrence.  Although  numerous  patients  suffering  from  the 
disease  come  to  England,  there  is  no  known  case  of  fresh 
infection  arising  under  natural  conditions. 

Agglutinative  Action  of  Serum. — The  blood  serum  of  patients 
suffering  from  Malta  fever  possesses  the  power  of  agglutinating 
the  micrococcus  melitensis  in  a  manner  analogous  to  what  has 
been  described  in  the  case  of  typhoid  fever.  The  reaction 
appears  comparatively  early,  often  about  the  fifth  day,  and  may 
be  present  for  a  considerable  time  after  recovery — sometimes 
for  more  than  a  year.  Distinct  agglutination  with  a  1  : 20 
dilution  of  the  serum  in  half  an  hour  may  be  taken  as  a  positive 
reaction,  sufficient  for  diagnosis.  The  reaction  is,  however, 
usually  given  by  much  higher  dilutions,  e.g.  1  : 500,  and  even 
higher.  It  is  to  be  noted  that  normal  serum  diluted  1  : 2  may 
produce  some  agglutination.  As  regards  relation  to  prognosis, 
the  observations  of  Birt  and  Lamb  and  of  Bassett-Smith  have 
given  results  analogous  to  those  obtained  in  typhoid  (p.  340). 

The  Commission  has  recently  found  that  vaccination  with 
dead  cultures  of  the  micrococcus  confers  a  certain  degree  of 
protection  amongst  those  exposed  to  the  disease.  As  a  rule  two 
injections  were  made,  200-300  million  cocci  being  the  dose  of 
the  first  injection,  and  about  400  million  of  the  second.  The 
use  of  vaccines  has  also  been  carried  out  in  the  treatment  of 
the  disease,  but  the  observations  are  not  sufficiently  numerous 
to  allow  a  definite  statement  to  be  made  as  to  its  value. 

Methods  of  Diagnosis. — During  life  the  readiest  means  of 
diagnosis  is  supplied  by  the  agglutinative  test  just  described 
(for  technique,  vide  p.  109). 

Cultures  are  most  easily  obtained  from  the  spleen  either 
during  life  or  post  mortem.  Inoculate  a  number  of  agar  tubes 
by  successive  strokes  and  incubate  at  37°  C.  Film  preparations 
should  also  be  made  from  the  spleen  pulp  and  stained  with 
carbol-thionin-blue  or  diluted  carbol-fuchsin  (1  :  10). 


YELLOW   FEVER  451 

YELLOW  FEVER. 

Yellow  fever  is  an  infectious  disease  which  is  endemic  in  the 
West  Indies,  in  Brazil,  in  Sierra  Leone  and  the  adjacent  parts 
of  West  Africa,  though  it  is  probable  that  it  was  from  the 
first-named  region  that  the  others  were  originally  infected. 
From  time  to  time  serious  outbreaks  occur,  during  which 
neighbouring  countries  also  suffer,  and  the  disease  may  be 
carried  to  other  parts  of  the  world.  In  this  way '  epidemics 
have  occurred  in  the  United  States,  in  Spain,  and  even  in 
England,  infection  usually  being  carried  by  cases  occurring 
among  the  crews  of  ships.  In  the  parts  where  it  is  endemic, 
though  usually  a  few  cases  may  occur  from  time  to  time,  there 
is  some  evidence  that  occasionally  the  disease  may  remain  in 
abeyance  for  many  years  and  then  originate  de  novo.  There  is, 
therefore,  reason  to  suspect  that  the  infective  agent  can  exist 
for  considerable  periods  outside  the  human  body.  It  is  possible, 
however,  that  continuity  may  be  maintained  by  the  occurrence 
of  a  mild  type  of  the  disease  which  may  be  grouped  with  the 
"  bilious  fevers  "  prevalent  in  yellow-fever  regions.  This  would 
explain  the  degree  of  immunity  which  is  shown  during  a  serious 
epidemic  by  the  older  immigrants. 

Great  variations  are  observed  in  the  clinical  types  under 
which  the  disease  presents  itself.  Usually  after  from  two  to 
six  days'  incubation  a  sudden  onset  in  the  form  of  a  rigor 
occurs:  The  temperature  rises  to  104°- 105°  F.  The  person  is 
livid,  with  outstanding  bloodshot  eyes.  There  are  present  great 
prostration,  pain  in  the  back,  and  vomiting,  at  first  of  mucus, 
later  of  bile.  The  urine  is  diminished  and  contains  albumin. 
About  the  fifth  day  an  apparent  improvement  takes  place,  and 
this  may  lead  on  to  recovery.  Frequently,  however,  the  remission, 
which  may  last  from  a  few  hours  to  two  days,  is  followed 
by  an  aggravation  of  all  the  symptoms.  The  temperature  rises, 
jaundice  is  observed,  haemorrhages  occur  from  all  the  mucous 
surfaces,  causing,  in  the  case  of  the  stomach,  the  "  black  vomit " 
— one  of  the  clinical  signs  of  the  disease  in  its  worst  form. 
Anuria,  coma,  and  cardiac  collapse  usher  in  a  fatal  issue.  The 
mortality  varies  in  different  epidemics  from  about  35  to  99 
per  cent  of  those  attacked.  Both  white  and  black  races  are 
susceptible,  but  those  who  have  resided  long  in  a  country  are 
less  susceptible  than  new  immigrants.  An  attack  of  the  disease 
usually  confers  complete  immunity  against  subsequent  infection. 

Post  mortem  the  stomach  is  found  in  a  state  of  acute  gastritis, 
and  contains  much  altered  blood  derived  from  hemorrhages 


452  YELLOW   FEVEK 

which  have  occurred  in  the  mucous  and  submucous  coats.  The 
intestine  may  be  normal,  but  is  often  congested  and  may  be 
ulcerated ;  the  mesenteric  glands  are  enlarged.  The  liver  is  in 
a  state  of  fatty  degeneration  of  greater  or  less  degree,  but  often 
resembling  the  condition  found  in  phosphorus  poisoning.  The 
kidneys  are  in  a  state  of  intense  glomerulo-nephritis,  with  fatty 
degeneration  of  the  epithelium.  There  is  congestion  of  the 
meninges,  especially  in  the  lumbar  region,  and  haemorrhages 
may  occur.  The  other  organs  do  not  show  much  change, 
though  small  hemorrhages  under  the  skin  and  into  all  the 
tissues  of  the  body  are  not  infrequent.  In  the  blood  a  feature 
is  the  excess  of  urea  present,  amounting,  it  may  be,  to  nearly 
4  per  cent. 

Etiology  of  Yellow  Fever. — Although  a  large  amount  of 
bacteriological  work  has  been  done  on  yellow  fever,  this  has 
now  chiefly  a  historical  interest,  as  it  is  now  known  that  the 
causal  agent  is  not  one  of  the  ordinary  bacteria,  but  belongs  to 
the  group  of  ultra-microscopic  organisms.1  A  mosquito  acts  as 
the  intermediate  host,  and  the  facts  detailed  below  point  to  the 
organism  passing  through  some  cycle  of  development  in  the 
body  of  the  insect.  The  analogy  of  malaria  makes  it  extremely 
probable  that  the  organism  is  a  protozoon,  but  as  this  has  not 
yet  been  completely  proved  we  have  not  felt  justified  in  altering 
the  position  of  the  disease  and  placing  it  amongst  the  protozoal 
infections.  As  bacteriological  work  led  up  to  the  establishment 
of  our  knowledge  regarding  the  nature  of  the  disease,  some 
reference  must  be  made  to  it. 

A  very  full  research  into  the  bacteriology  of  yellow  fever 
was  that  of  Sternberg,  the  result  of  which  was  that  of  the 
varied  organisms  isolated,  one  which  he  called  the  bacillus  x 
appeared  possibly  to  have  some  relationship  to  the  disease. 
Sanarelli  in  1897  obtained  cultures  of  an  organism  which  he 
called  bacillus  icteroides,  and  which  he  considered  to  be  the 
cause  of  yellow  fever ;  it  is  probably  identical  with  the  bacillus 
x  of  Sternberg.  Subsequent  observations  made  by  others 
gave  conflicting  results,  some  finding  this  bacillus,  others 
failing  to  do  so.  The  bacillus  icteroides,  as  described  by 
Sanarelli,  belongs  to  the  paratyphoid  group,  possessing  lateral 
flagella,  growing  on  gelatin  without  liquefaction,  and  fermenting 
glucose  but  not  lactose.  Reed  and  Carroll  found  that  it  was 
practically  identical  with  the  bacillus  of  swine  cholera.  It 

1  In  several  diseases  the  existence  of  such  causal  factors  is  suspected. 
Other  examples  are  foot  and  mouth  disease,  South  African  horse-sickness,  and 
the  contagious  pleuro-pneumonia  of  cattle. 


ETIOLOGY   OF  YELLOW   FEVER  453 

must  now  be  considered  merely  as  an  organism  which  may 
occur  in  the  organs  and  tissues  in  yellow  fever  as  a  secondary 
infection,  but  without  any  etiological  significance. 

The  facts  of  importance  which  have  been  established 
regarding  the  etiology  of  the  disease  are  due  to  the  labours  of 
the  United  States  Army  Commission,  which  began  its  work  in 
1900.  The  members  of  the  Commission  first  directed  their 
inquiries  towards  determining  whether  the  bacillus  icteroides 
was  present  in  the  blood  during  life,  and  a  series  of  cases  were 
investigated  bacteriologically,  with  entirely  negative  results  in 
each  instance.  They  then  resolved  to  test  the  hypothesis  of 
Dr.  Carlos  Finlay  of  Havana,  promulgated  several  years  pre- 
viously, that  the  disease  was  carried  by  mosquitoes.  Selecting 
mosquitoes  which  they  reared  from  eggs,  they  allowed  them  to 
bite  yellow  fever  patients  and  then  to  bite  healthy  men.  Of 
several  experiments  of  this  nature  two  were  successful  in  the 
first  instance,  the  first  individual  to  be  infected  in  this  way 
being  Dr.  James  Carroll,  a  member  of  the  Commission,  who 
passed  through  a  severe  attack  of  typical  yellow  fever.  Experi- 
ments were  then  performed  on  a  larger  scale,  with  completely 
confirmatory  results,  as  to  the  conveyance  of  the  disease  by 
mosquitoes.  Of  twelve  non-immunes  living  under  circumstances 
which  excluded  natural  means  of  infection,  ten  contracted 
yellow  fever  after  having  been  bitten  by  mosquitoes  which  had 
previously  bitten  yellow  fever  patients ;  happily  all  of  these 
recovered.  Two  of  the  men  who  were  thus  infected  had  been 
previously  exposed  to  contact  with  fomites  from  yellow  fever 
patients  without  .results.  These  results  were  confirmed  by 
Guiteras,  whose  investigations  were  carried  out  along  similar 
lines;  of  seventeen  individuals  bitten  by  infected  mosquitoes, 
eight  took  yellow  fever,  and  three  of  these  died. 

The  species  of  mosquito  used  by  the  American  Commission 
was  the  Stegomyia  fasciata,  and  up  to  the  present  time  no  other 
species  has  been  found  capable  of  carrying  the  infection.  It  has 
also  been  determined  that  a  certain  period  must  elapse  after  the 
insect  has  bitten  a  yellow  fever  patient  before  it  becomes  infec- 
tive to  another  subject.  In  summer  weather  this  period  is  about 
twelve  days ;  at  a  lower  temperature  somewhat  longer.  This 
probably  means  that,  as  in  the  case  of  malaria,  the  parasite  must 
pass  through  certain  stages  of  development  before  it  reaches  the 
salivary  gland  and  is  thus  in  a  position  to  be  transferred  to  a  fresh 
subject.  Infected  mosquitoes,  however,  retain  the  power  of 
infection  for  a  considerable  time  afterwards,  probably  as  long  as 
sixty  days.  It  has  also  been  shown  that  mosquitoes  may  become 


454  YELLOW  FEVER 

infective  after  biting  a  patient  on  the  first,  second,  or  third  day 
of  the  disease,  but  at  a  later  period  the  results  are  usually 
negative,  apparently  because  the  virus  is  no  longer  present  in 
the  blood. 

Interesting  results  were  also  obtained  with  regard  to  the 
communication  of  the  disease  directly  from  patient  to  patient, 
the  conclusion  arrived  at,  after  careful  experiments,  being  that 
the  disease  cannot  be  transferred  in  this  way,  even  when  the 
contact  is  of  a  close  character.  In  a  specially  constructed  house 
seven  men  were  exposed  to  the  most  intimate  contact  with  the 
fomites  of  yellow  fever  patients  for  a  period  of  twenty  days  each, 
the  soiled  garments  worn  by  the  patients  being  in  some  cases 
actually  slept  in  by  these  men  ;  the  result  was  that  not  one  of 
them  thus  exposed  contracted  the  disease.  The  conclusions  on 
this  point  have  been  subsequently  confirmed  by  other  workers. 

The  American  Commission  also  found  it  possible  to  transmit 
yellow  fever  to  a  healthy  man  by  injecting  small  quantities  of 
blood  or  of  serum  taken  from  a  yellow  fever  patient  at  any 
period  up  till  the  third  day  of  the  disease.  The  period  of 
incubation  in  this  case  is  somewhat  shorter  than  when  the  disease 
is  conveyed  by  the  bite  of  mosquitoes,  the  average  duration  in 
the  former  case  being  about  three  days,  and  in  the  latter  about 
four  days,  though  these  times  may  be  considerably  exceeded. 
It  is  also  interesting  to  know  that  in  these  experimental  injec- 
tions the  blood  or  serum  used  was  found  to  be  free  from  bacteria. 
Up  till  the  present  time,  we  know  of  only  these  two  methods  of 
infection,  namely,  indirectly  by  the  bite  of  a  mosquito  infected 
with  the  yellow  fever  germ,  or  directly  by  the  injection  of  some 
of  the  blood  from  a  yellow  fever  patient.  In  these  respects 
there  is  a  striking  similarity  to  what  has  been  established  in  the 
case  of  malarial  fever. 

Experiments  with  regard  to  the  nature  of  the  yellow  fever 
organism  were  carried  out  by  Reed  and  Carroll,  and  interest- 
ing results  were  obtained.  They  found  that  the  organism  of 
the  disease  was  very  easily  killed  by  heat,  as  blood  from  a 
yellow  fever  patient  lost  its  infective  power  on  being  heated 
to  55°  C.  for  ten  minutes.  On  the  other  hand,  blood  or  serum 
was  found  to  be  still  infective  after  having  been  passed  through 
a  Berkefeld  filter.  This  has  been  confirmed  by  the  French 
Commission,  with  the  additional  result  that  the  virus  passes 
through  a  Chamberland  F  filter,  but  not  through  a  Chamber- 
land  B.  These  facts  would  show  that  the  parasite  is  of 
extremely  minute  size,  and  apparently  belongs  to  the  group  of 
ultra  -  microscopic  organisms.  Up  till  the  present  time  all 


ETIOLOGY   OF   YELLOW   FEVER  455 

attempts  to  find  by  microscopic  examination  the  yellow  fever 
parasite,  either  in  the  blood  of  patients  suffering  from  the 
disease,  or  in  the  tissues  of  infective  mosquitoes,  have  been 
attended  with  negative  results. 

Though  nothing  has  been  determined  regarding  the  actual 
nature  of  the  virus,  yet  the  results  already  obtained  have 
supplied  the  basis  for  preventive  measures  against  the  disease, 
these  being  directed  towards  the  destruction  of  mosquitoes  and 
the  protection  of  those  suffering  from  yellow  fever,  and  also  the 
healthy,  against  the  bites  of  these  insects.  Already  a  striking 
degree  of  success  has  been  obtained  in  Havana.  Such  measures 
came  into  force  in  February  1901,  and  in  ninety  days  the  town 
was  free  of  yellow  fever  and  for  fifty-four  days  later  no  new 
cases  occurred;  and  although  subsequently  the  disease  was 
reintroduced  into  the  town,  no  difficulty  was  experienced  in 
stamping  it  out  by  the  same  measures.  In  recent  years  the 
results  have  also  been  highly  gratifying,  and  the  disease  may 
be  said  to  be  practically  eradicated  from  Havana.  In  other 
places  also  successful  results  have  been  obtained,  and  epidemics 
would  appear  to  be  now  under  control  if  the  proper  measures 
are  taken. 


CHAPTER  XIX. 

IMMUNITY. 

Introductory. — By  immunity  is  meant  non-susceptibility  to  a 
given  disease  or  to  a  given  organism,  either  under  natural 
conditions  or  under  conditions  experimentally  produced.  The 
term  is  also  used  in  relation  to  the  toxins  of  an  organism. 
Immunity  may  be  possessed  by  an  animal  naturally,  and  is  then 
usually  called  natural  immunity,  or  it  may  be  acquired  by  an 
animal  either  by  passing  through  an  attack  of  the  disease,  or  by 
artificial  means  of  inoculation.  It  is  to  be  noted  that  man  and 
the  lower  animals  may  be  exempt  from  certain  diseases  under 
natural  conditions,  and  yet  the  causal  organisms  of  these  diseases 
may  produce  pathogenic  effects  when  injected  in  sufficient 
quantity.  Immunity  is,  in  fact,  of  very  varying  degrees,  and 
accordingly  the  use  of  the  term  has  a  correspondingly  relative 
significance.  This  is  not  only  true  of  infection  by  bacteria,  but 
of  toxins  B!SO  : — when  the  resistance  of  an  animal  to  these  is  of 
high  degree,  the  resistance  may  in  certain  cases  be  overcome  by 
a  very  large  dose  of  the  toxic  agent.  For  example,  the  common 
fowl  may  be  able  to  resist  as  much  as  20  c.c.  of  powerful  tetanus 
toxin,  but  on  this  amount  being  exceeded  may  be  affected  by 
tetanic  spasms  (Klemperer).  On  the  other  hand,  in  cases  where 
the  natural  powers  of  resistance  are  very  high,  these  can  be  still 
further  exalted  by  artificial  means,  that  is,  the  natural  immunity 
may  be  artificially  intensified. 

Acquired  Immunity  in  the  Human  Subject. — The  following 
facts  are  supplied  by  a  study  of  the  natural  diseases  which  affect 
the  human  subject.  First,  in  the  case  of  certain  diseases,  one 
attack  protects  against  another  for  many  years,  sometimes 
practically  for  a  lifetime,  e.g.  smallpox,  typhoid,  scarlet  fever, 
etc.  Secondly,  in  the  case  of  other  diseases,  e.g.  erysipelas, 
diphtheria,  influenza,  and  pneumonia,  a  patient  may  suffer  from 
several  attacks.  In  the  case  of  the  diseases  of  the  second  group, 

456 


ARTIFICIAL   IMMUNITY  457 

however,  experimental  research  has  shown  that  in  many  of  them 
a  certain  degree  of  immunity  does  follow ;  and,  though  we 
cannot  definitely  state  it  as  a  universal  law,  it  must  be  con- 
sidered highly  probable  that  the  passing  through  an  attack  of  an 
acute  disease  produced  by  an  organism,  confers  immunity  for  a 
longer  or  shorter  period.  The  immunity  is  not,  however,  to  be 
regarded  as  the  result  of  the  disease  per  se,  but  of  the  bacterial 
products  introduced  into  the  system ;  as  will  be  shown  below, 
by  suitable  gradation  of  the  doses  of  such  products,  or  by  the 
use  of  weakened  toxins,  a  high  degree  of  immunity  may  be 
attained  without  the  occurrence  of  any  symptoms  whatever. 

The  facts  known  regarding  vaccination  and  smallpox 
exemplify  another  principle.  We  may  take  it  as  practically 
proved  that  vaccinia  is  variola  or  smallpox  in  the  cow,  and  that 
when  vaccination  is  performed,  the  patient  is  inoculated  with  a 
modified  variola  (vide  Smallpox,  in  Appendix).  Vaccination 
produces  certain  pathogenic  effects  which  are  of  trifling  degree 
as  compared  with  those  of  smallpox,  and  we  find  that  the  degree 
of  protection  is  less  complete  and  lasts  a  shorter  time  than  that 
produced  by  the  natural  disease.  Again,  inoculation  with  lymph 
from  a  smallpox  pustule  produces  a  form  of  smallpox  less 
severe  than  the  natural  disease  but  a  much  more  severe  con- 
dition than  that  produced  by  vaccination,  and  it  is  found  that 
the  degree  of  protection  or  immunity  resulting  occupies  an  inter- 
mediate position. 

Immunity  and  Recovery  from  Disease. — Recovery  from  an 
acute  infective  disease  shows  that  in  natural  conditions  the  virus 
may  be  exhausted  after  a  time,  the  period  of  time  varying  in 
different  diseases.  How  this  is  accomplished  we  do  not  yet 
fully  know,  but  it  has  been  found  in  the  case  of  diphtheria, 
typhoid,  cholera,  pneumonia,  etc.,  that  in  the  course  of  the 
disease  certain  substances  (called  by  German  writers  Antikorper) 
appear  in  the  blood,  which  are  antagonistic  either  to  the  toxin 
or  to  the  vital  activity  of  the  organism.  In  such  cases  a  process 
of  immunisation  would  appear  to  be  going  on  during  the  pro- 
gress of  the  disease,  and  when  this  immunisation  has  reached  a 
certain  height,  the  disease  naturally  comes  to  an  end.  It  can- 
not, however,  be  said  as  yet  that  such  antagonistic  substances 
are  developed  in  all  cases ;  though  the  results  already  obtained 
make  this  probable. 

AKTIFICIAL  IMMUNITY 

Varieties. — According  to  the  means  by  which  it  is  produced, 
immunity  may  be  said  to  be  of  two  kinds,  to  which  the  terms 


458  IMMUNITY 

active  and  passive  are  generally  applied,  or  we  may  speak  of 
immunity  directly,  or  indirectly,  produced.  We  shall  first  give 
an  account  of  the  established  facts,  and  afterwards  discuss  some 
of  the  theories  which  have  been  brought  forward  in  explanation 
of  these  facts. 

Active  immunity  is  obtained  by  (a)  injections  of  the  organisms 
either  in  an  attenuated  condition  or  in  sub-lethal  doses,  or  (b) 
by  sub-lethal  doses  of  their  products,  i.e.  of  their  "  toxins,"  the 
word  being  used  in  the  widest  sense.  By  repeated  injections 
at  suitable  intervals  the  dose  of  organisms  or  of  the  products 
can  be  gradually  increased  ;  or,  what  practically  amounts  to  the 
same,  an  organism  of  greater  virulence  or  a  toxin  of  greater 
strength  may  be  used.  A  proportionate  degree  of  resistance  or 
immunity  can  thus  be  developed,  which  degree  in  course  of  time 
may  reach  a  very  high  level.  Such  methods  constitute  the 
means  of  preventive  inoculation  or  vaccination.  Immunity  of 
this  kind  is  comparatively  slowly  produced  and  lasts  a  consider- 
able time,  the  duration  varying  in  different  cases. 

Passive  immunity  depends  upon  the  fact  that  if  an  animal 
be  immunised  to  a  very  high  degree  by  the  previous  method,  its 
serum  may  have  distinctly  antagonistic  or  neutralising  effects 
when  injected  into  another  animal  along  with  the  organisms,  or 
with  their  products,  as  the  case  may  be.  Such  a  serum,  generally 
known  as  an  anti-serum,  may  exert  its  effects  if  introduced  into 
an  animal  at  the  same  time  as  infection  occurs  or  even  a  short 
time  afterwards ;  it  can,  therefore,  be  employed  as  a  curative 
agent.  The  serum  is  also  preventive,  i.e.  protects  an  animal 
from  subsequent  infection,  but  the  immunity  thus  conferred 
lasts  a  comparatively  short  time.  These  facts  form  the  basis  of 
serum  therapeutics.  When  such  a  serum  has  the  power  of 
neutralising  a  toxin  it  is  called  antitoxic ;  when,  with  little  or 
no  antitoxic  power,  it  protects  against  the  living  bacterium  in  a 
virulent  condition,  it  is  called  antimicrobic  or  antibacterial  (vide 
infra). 

In  the  accompanying  table  a  sketch  of  the  chief  methods  by 
which  an  immunity  may  be  artificially  produced  is  given.  It 
has  been  arranged  merely  for  purposes  of  convenience  and  to 
aid  subsequent  description ;  the  principles  underlying  all  the 
methods  are  the  same. 

ARTIFICIAL  IMMUNITY. 

A.  Active  Immunity — i.e.  produced  in  an  animal  by  an  in- 
jection, or  by  a  series  of  injections,  of  non-lethal  doses  of 
an  organism  or  its  toxins. 


ARTIFICIAL   IMMUNITY  459 

1 .  By  injection  of  the  living  organisms. 

(a)  Attenuated  in  various  ways.     Examples  : — 

(1)  By  growing  in  the  presence  of  oxygen,  or  in  a 

current  of  air. 

(2)  By  passing  through   the    tissues   of   one  species 

of   animal  (becomes   attenuated   for   another 
species). 

(3)  By  growing  at  abnormal  temperatures,  etc. 

(4)  By  growing  in  the  presence  of  weak  antiseptics,  or 

by  injecting  the  latter  along  with  the  organism, 
etc. 

(b)  In  a  virulent  condition,  in  non-lethal  doses. 

2.  By  injection  of  the  dead  organisms. 

3.  By  injection  of  filtered  bacterial  cultures,  i.e.  toxins  ;  or  of 

chemical  substances  derived  from  such  filtrates. 
These  methods  may  also  be  combined  in  various  ways. 

B.  Passive  Immunity,  i.e.  produced  in  one  animal  by  injection 
of  the  serum  of  another  animal  highly  immunised  by  the 
methods  of  A. 

1.  By  antitoxic  serum,  i.e.  the  serum  of  an  animal  highly 

immunised  against  a  particular  toxin. 

2.  By  antibacterial  serum,  i.e.  the  serum  of  an  Animal  highly 

immunised  against  a  particular  bacterium  in  the  living 
and  virulent  condition. 

A.  Active  Immunity. 

1.  By  Living  Cultures. — (a)  Attenuated. — In  the  earlier 
work  on  immunity  in  the  case  of  anthrax,  chicken  cholera,  swine 
plague,  etc.,  the  investigators  had  to  deal  with  organisms  of 
high  virulence,  which  had  accordingly  to  be  reduced  before  the 
organisms  could  be  injected  in  the  living  state.  It  is  now  found 
most  convenient  as  a  rule  to  start  the  process  of  active  immunisa- 
tion with  the  injection  of  dead  cultures.  The  principle  is  the 
same  as  that  of  vaccination,  and  both  attenuated  cultures  and 
also  the  dead  cultures  used  for  injection  are  often  spoken  of  as 
vaccines.  The  virulence  of  an  organism  may  be  diminished  in 
various  ways,  of  which  the  following  examples  may  be  given. 

(1)  In  the  first  place,  practically  every  organism  when  culti- 
vated for  some  time  outside  the  body,  loses  its  virulence,  and 
in  the  case  of  some  this  is  very  marked  indeed,  e.g.  the  pneumo- 


460  IMMUNITY 

coccus.  Pasteur  found  in  the  case  of  chicken  cholera,  that 
when  cultures  were  kept  for  a  time  in  ordinary  conditions,  they 
gradually  lost  their  virulence,  and  that  when  sub-cultures  were 
made  the  diminished  virulence  persisted.  Such  attenuated 
cultures  could  be  used  for  protective  inocula'tion.  He  considered 
the  loss  of  virulence  to  be  due  to  the  action  of  the  oxygen  of 
the  air,  as  he  found  that  in  tubes  sealed  in  the  absence  of  oxygen 
the  virulence  was  not  lost.  Haffkine  attenuated  cultures  of  the 
cholera  spirillum  by  growing  them  in  a  current  of  air  (p.  412). 

(2)  The  virulence  of  an  organism  for  a  particular  animal  may 
be  lessened  by  passing  the  organism  through  the  body  of  another 
animal.     Duguid  and  Burdon  Sanderson  found  that  the  virulence 
of  the  anthrax  bacillus  for  bovine  animals  was  lessened  by  its  being 
passed  through  guinea-pigs,  the  disease  produced  in  the  ox  by 
inoculation  from  the  guinea-pig  being  a  non-fatal  one.     This  dis- 
covery was  confirmed  by  Greenfield,  who  showed  that  the  bacilli 
cultivated  from  guinea-pigs  preserved  their  property  in  cultures, 
and  could  therefore  be  used  for  protective  inoculation  of  cattle. 
A  similar  principle  was  applied  in  the  case  of  swine  plague  by 
Pasteur,  who  found  that  if  the  organism  producing  this  disease 
was  inoculated  from  rabbit  to  rabbit,  its  virulence  was  increased 
for  rabbits  but  was  diminished  for  pigs.     The  method  of  vaccina- 
tion against  smallpox  depends  upon  the  same  principle.     There 
is  also  evidence  to   show  that   the  virulence  of   the    tubercle 
bacillus  becomes  modified   according  to  its  host,   being   often 
diminished  for  other  animals. 

(3)  Many  organisms  become  diminished  in  virulence  when 
grown   at   an  abnormally  high   temperature.     The   method   of 
Pasteur,  already  described  (p.  314),  for  producing  immunity  in 
sheep  against  anthrax  bacilli,  depends  upon  this  fact.     A  virulent 
organism  may  also  be  attenuated  by  being  exposed  to  an  elevated 
temperature  which  is  insufficient  to  kill   it,  as  was  found  by 
Toussaint  in  the  case  of  anthrax. 

(4)  Still  another  method  may  be  mentioned,    namely,   the 
attenuation  of  the  virulence  by  growing  the  organism  in  the 
presence   of   weak  antiseptics.     Chamberland    and    Roux,    for 
example,    succeeded   in    attenuating   the   anthrax    bacillus    by 
growing    it    in    a    medium    containing    carbolic    acid    in    the 
proportion  of  1  : 600. 

These  examples  will  serve  to  show  the  principles  underlying 
attenuation  of  the  virulence  of  an  organism.  There  are,  how- 
ever, still  other  methods,  most  of  which  consist  in  growing  the 
organism  in  conditions  somewhat  unfavourable  to  its  growth,  e.g. 
under  compressed  air,  etc. 


BY   LIVING   CULTURES  461 

(5)  Immunity  by  living  Virulent  Cultures  in  Non-lethal 
Doses. — Immunity  may  also  be  produced  by  employing  virulent 
cultures  in  small,  that  is  non-lethal,  doses.  In  subsequent 
inoculations  the  doses  may  be  increased  in  amount.  For  example, 
immunity  may  thus  be  obtained  in  rabbits  against  the  bacillus 
pyocyaneus.  Such  a  method,  however,  has  had  a  limited 
application  in  the  case  of  virulent  organisms,  as  it  has  been 
found  more  convenient  to  commence  the  process  by  attenuated 
cultures,  and  then  to  continue  with  living  cultures. 

Exaltation  of  the  Virulence.  —  The  converse  process  to 
.attenuation,  i.e.  the  exaltation  of  the  virulence,  is  obtained 
chiefly  by  the  method  of  cultivating  the  organism  from  animal 
to  animal — the  method  of  passage  discovered  by  Pasteur  (first, 
we  believe,  in  the  case  of  an  organism  obtained  from  the  saliva 
in  hydrophobia,  though  having  no  causal  relationship  to  that 
disease).  This  is  most  conveniently  done  by  intraperitoneal 
injections,  as  there  is  less  risk  of  contamination.  The  organisms 
in  the  peritoneal  fluid  may  be  used  for  the  subsequent  injection, 
or  a  culture  may  be  made  between  each  inoculation.  The 
virulence  of  a  great  number  of  organisms  can  be  increased  in 
this  way,  the  animals  most  frequently  used  being  rabbits  and 
guinea-pigs.  This  method  can  be  applied  to  the  organisms  of 
typhoid,  cholera,  pneumonia,  to  streptococci  and  staphylococci, 
and  in  fact  to  those  organisms  generally  which  invade  the 
tissues. 

The  virulence  of  an  organism,  especially  when  in  a  relatively 
attenuated  condition,  can  also  be  raised  by  injecting  along  with 
it  a  quantity  of  a  culture  of  another  organism  either  in  the  living 
or  dead  condition.  A  few  examples  may  be  mentioned.  An 
attenuated  diphtheria  culture  may  have  its  virulence  raised  by 
being  injected  into  an  animal  along  with  the  streptococcus 
pyogenes ;  an  attenuated  culture  of  the  bacillus  of  malignant 
redema  by  being  injected  with  the"  bacillus  prodigiosus ;  an 
attenuated  streptococcus  by  being  injected  with  the  bacillus  coli, 
etc.  A  culture  of  the  typhoid  bacillus  may  be  increased  in 
virulence,  as  already  stated,  by  being  injected  along  with  a  dead 
culture  of  the  bacillus  coli.  In  such  cases  the  accompanying  in- 
jection enables  the  attenuated  organism  to  gain  a  foothold  in  the 
tissues,  and  it  may  be  stated  as  a  general  rule  that  the  virulence 
of  an  organism  for  a  particular  animal  is  raised  by  its  growing 
in  the  tissues  of  that  animal. 

Combination  of  Methods. — The  above  methods  may  be  com- 
bined in  various  ways.  By  repeated  injections  of  cultures  at 
first  in  the  dead  condition,  then  living  and  attenuated  and 


462  IMMUNITY 

afterwards  more  virulent,  and  by  increasing  the  doses,  a  high 
degree  of  immunity  may  be  obtained. 

2.  Immunity  by  Dead  Cultures  of  Bacteria. — In  some  cases 
a  high  degree  of  immunity  against  infection  by  a  given  microbe 
may  be  developed  by  repeated  and  gradually  increasing  doses 
of  the  dead  cultures,  the  cultures  being  killed  sometimes  by 
heat,  sometimes  by  exposure  to  the  vapour  of  chloroform.     In 
this  method  the  so-called  endotoxins  will  be  injected  along  with 
the  other  substances  in  the  bacterial  protoplasm,  but  the  resulting 
immunity  is  chiefly  directed  against  the   vital  activity  of  the 
organisms — is  antibacterial  rather  than  antitoxic  (vide  infra). 
The  cultures  when  dead  produce,  of  course,  less  effect  than  when 
living,  and  this  method  may  be  conveniently  used  in  the  initial 
stages  of  active  immunisation,   to  be  afterwards  followed    by 
injections  of  the  living  cultures.     The  method    is  extensively 
used  for  experimental  purposes,   and  is  that  adopted  in  anti- 
plague  and  anti-typhoid  inoculations. 

3.  Immunity  by  the    Separated    Bacterial    Products   or 
Toxins. — The  organisms  in  a  virulent  condition  are  grown  in 
a  fluid  medium  for  a  certain  time,  and  the  fluid  is  then  filtered 
through  a  Chamberland  or  other  porcelain  filter.     The  filtrate 
contains  the  toxins,  and  it  may  be  used  unaltered,  or  may  be 
reduced    in   bulk   by   evaporation,    or   may   be   evaporated   to 
dry  ness.     The  process  of  immunisation  by  the  toxin  is  started 
by  small  non-lethal  doses  of  the  strong  toxin,  or  by  larger  doses 
of  toxin  the  power  of  which  has  been  weakened  by  various 
methods    (vide   infra}.      Afterwards    the    doses    are   gradually 
increased.     This  method  was  carried  out  with  a  great  degree 
of  success  in  the  case  of  diphtheria,  tetanus,  malignant  osdema, 
etc.     It  appears  capable  of  general  application  in  the  case  of 
organisms  where  it  is  possible  to  get  an  active  toxin  from  the 
filtered  cultures.     It  has  also  been  applied  in  the  case  of  snake 
poisons  by  Calmette  and  by  Fraser,  and  a  high  degree  of  im- 
munity has  been  produced. 

Immunity  may  also  be  obtained  by  means  of  certain  chemical 
substances  separated  from  filtered  bacterial  cultures,  though 
these  substances  are  generally  in  a  more  or  less  impure 
condition.  Hankin  was  the  first  to  obtain  this  result  by  means 
of  an  albumose  separated  from  anthrax  cultures. 

The  following  may  be  mentioned  as  some  of  the  most 
important  examples  of  the  practical  application  of  the  principles 
of  active  immunity,  i.e.  of  protective  inoculation  : — (1)  Inocula- 
tion of  sheep  and  oxen  against  anthrax  (Pasteur)  (p.  314) ;  (2) 
Jennerian  vaccination  against  smallpox  (p.  503) ;  (3)  Anti- 


BY   BACTERIAL   PRODUCTS   OR   TOXINS       463 

cholera  inoculation  (Haffkine)  (p.  412);  (4)  Anti-plague 
inoculation  (Haffkine)  (p.  434) ;  (5)  Anti-typhoid  inoculation 
(Wright  and  Semple)  (p.  343) ;  (6)  Pasteur's  method  of  inocu- 
lation against  hydrophobia,  which  involves  essentially  the  same 
principles  (p.  516). 

Vaccines  as  a  Method  of  Treatment. — Up  till  recently  the 
principles  of  active  immunity  had  not  been  directly  applied  in 
the  treatment  of  an  existing  disease  except  in  the  case  of 
tuberculosis.  The  work  of  Wright,  however,  shows  that  active 
immunisation  in  such  circumstances  is  not  only  possible,  but  is 
also  probably  capable  of  wide  application.  From  his  study  of 
the  part  played  by  phagocytosis  in  the  successful  combat  of 
bacteria  by  the  body,  he  was  led  to  advocate  the  treatment  of 
bacterial  infections  by  carrying  on  an  active  immunisation 
against  the  causal  agents  by  the  injection  of  dead  cultures  of 
the  latter.  The  justification-  for  such  a  procedure  lies  in  his 
contention  that  in  many  cases  infections  are  to  be  looked  on  as 
practically  entirely  localised,  e.g.  the  cases  of  an  acne  pustule, 
or  a  boil.  The  reason  for  the  local  growth  of  bacteria  in  the 
part  of  the  body  affected  is  that  there  is  for  unknown  causes  a 
deficiency  of  the  opsonic  power  (vide  p.  483)  of  the  body  fluids, 
which  is  essential  for  the  phagocytosis  of  the  invading  bacteria. 
Still  more  marked  in  such  cases  is  the  deficiency  in  the  opsonic 
qualities  of  the  fluids  in  the  actual  site  of  infection.  Any 
procedure  which  will  raise  the  opsonic  power  of  the  body  fluids 
as  a  whole,  and  therefore  of  the  fluids  in  the  focus  of  infection, 
will  aid  the  destruction  of  the  bacteria  by  sensitising  them  to 
phagocytic  action.  Such  a  procedure  is  found  in  the  active 
immunisation  which  results  from  the  injection  of  a  vaccine 
consisting  of  a  dead  culture  of  the  causal  bacteria.  The  appli- 
cation of  a  vaccine  of  this  kind  must,  however,  be  controlled  by 
constant  observation  of  the  opsonic  index  of  the  patient's  serum 
during  the  treatment.  When  a  local  infection  occurs  the  general 
opsonic  index  is  usually  found  to  be  below  unity.  If  dead 
bacteria  be  injected  into  the  individual  there  may  occur  during 
the  following  few  days  a  further  fall  in  the  opsonic  index, — 
what  Wright  calls  the  occurrence  of  a  negative  phase.  In  a  case 
where  the  treatment  is  successful,  this  negative  phase  is  succeeded 
by  a  rise  in  the  opsonic  index  above  its  original  level, — ocurrence 
of  positive  phase, — and  with  this  reaction  there  is  an  improve- 
ment in  the  local  condition.  Usually  in  such  cases  repeated 
injections  are  required  to  effect  a  cure,  and  the  important  point, 
according  to  Wright,  is  to  avoid  giving  an  injection  when  a 
negative  phase  is  in  progress.  If  this  point  is  not  attended  to 


464  IMMUNITY 

only  an  aggravation   of   symptoms   is    to    be  looked  for  (vide 
pp.  194,  261). 

With  regard  to  the  details  of  the  preparation  of  the  vaccines, 
an  agar  culture  is  taken,  the  growth  removed  into  normal  saline 
and  killed  by  steaming  for  a  sufficient  time — say  1J  hours. 
The  efficiency  of  the  sterilisation  is  tested  by  inoculating  tubes 
of  appropriate  media.  The  strength  of  the  emulsion  is  then 
estimated  by  the  method  of  counting  dead  bacteria  described  on 
p.  67.  The  number  of  bacteria  employed  for  a  vaccination  is 
usually  from  250,000,000  to  500,000,000,  and  in  the  details  of 
the  measurement  of  this  quantity  and  in  its  injection,  every 
aseptic  precaution  must,  of  course,  be  adopted.  Such  vaccines 
have  been  used  extensively  in  the  treatment  of  acne,  boils, 
sycosis,  infections  of  the  genito-urinary  tract  by  the  b.  coli, 
infections  of  joints  by  the  gonococcus,  and  in  many  cases  con- 
siderable success  has  followed  the  treatment. 

Active  Immunity  by  Feeding. — Ehrlich  found  that  mice 
could  be  gradually  immunised  against  ricin  and  abrin  by  feeding 
them  with  increasing  quantities  of  these  substances  (vide  p.  169). 
In  the  course  of  some  weeks'  treatment  in  this  way  the  resulting 
immunity  was  of  so  high  a  degree  that  the  animals  could  tolerate 
on  subcutaneous  inoculation  400  times  the  dose  originally  fatal. 
Fraser  also  found  in  the  case  of  snake  venom  that  rabbits  could 
be  immunised,  by  feeding  with  the  poison,  against  several  times 
the  lethal  dose  of  venom  injected  into  the  tissues. 

By>  feeding  animals  with  dead  cultures  of  bacteria  or  with 
their  separated  toxins,  a  degree  of  immunity  may  in  some  cases 
be  gradually  developed.  But  this  method  is  so  much  less  certain 
in  results,  and  so  much  more  tedious  than  the  others,  that  it  has 
obtained  no  practical  applications. 

Active  immunity  of  high  degree  developed  by  the  methods 
described  may  be  regarded  as  specific,  that  is,  is  exerted  only 
towards  the  organism  or  toxin  by  means  of  which  it  has  been 
produced.  A  certain  degree  of  immunity,  or  rather  of  increased 
general  resistance  of  parts  of  the  body  (for  example  the  peri- 
toneum), can,  however,  be  produced  by  the  injection  of  various 
substances — bouillon,  blood  serum,  solution  of  nuclein,  etc. 
(Issaeff).  Also  increased  resistance  to  one  organism  can  be  thus 
produced  by  injections  of  another  organism.  Immunity  of  this 
kind,  however,  never  reaches  a  high  degree. 

B.  Passive  Immunity. 

Action  of  the  Serum  of  Highly-Immunised  Animals. — 1. 

The  serum  of  an  animal  A,  treated  by  repeated  and  gradually 


PASSIVE   IMMUNITY  465 

increased  doses  of  a  toxin  of  a  particular  microbe,  may  protect 
an  animal  B  against  a  certain  amount  of  the  same  toxin  when 
injected  along  with  thfe  latter,  or  a  short  time  before  it.  As 
would  be  expected,  it  has  less  effect  when  injected  some  time 
afterwards,  but  even  then  within  certain  limits  it  has  a  degree 
of  curative  or  palliative  power.  Seeing  that  the  serum  of  animal 
A  appears  to  neutralise  the  toxin,  the  term  antitoxic  has  been 
applied  to  it. 

2.  The  serum  of  an  animal  A,  highly  immunised  against  a 
bacterium  by  repeated  and  gradually  increasing  doses  of  the 
organism,  may  protect  an  animal  B  against  an  infection  by  the 
living  organism  when  injected  under  conditions  similar  to  the 
above.  This  serum  is  therefore  antimicrobic,  or  antibacterial, 
i.e.  preventive  against  invasion  by  a  particular  organism.  (In 
addition  to  the  preventive  or  protective  action  in  vivo,  such  a 
serum  may  exert  certain  recognisable  effects  on  the  corresponding 
organism  in  vitro.  Thus  (a)  it  may  lead  to  the  death  or  solution 
of  the  organism — bactericidal  or  lysogenic  action;  (b)  it  may 
produce  an  increased  susceptibility  to  ingestion  by  phagocytes — 
opsonic  action ;  (c)  it  may  lead  to  the  clumping  of  the  organism 
— agglutinative  action.) 

These  two  kinds  of  anti-sera  —  antitoxic  and  antibacterial  — 
exert  their  effect  when  injected  along  with  the  toxin  or  organism 
respectively  or  some  time  previously ;  as  would  be  expected, 
they  have  less  effect  when  injected  some  time  afterwards,  though 
even  then  they  may  have  a  certain  degree  of  curative  or  palliative 
power..  The  two  properties,  antitoxic  and  antibacterial,  are  essen- 
tially different  in  kind,  the  former  leading  to  a  neutralisation  of 
the  toxin,  the  latter  to  some  alteration  in  the  vital  activity  of 
the  bacterium ;  in  other  words,  the  point  of  attack  in  the  case 
of  the  two  sera  is  different.  A  serum  may,  however,  possess 
both  properties  in  varying  degree.  The  fundamental  fact  in 
passive  immunity,  viz.  that  immunity  can  be  transferred  to 
another  animal,  shows  that  the  serum  in  question  differs  from 
the  serum  of  a  normal  animal  in  containing  antagonistic  sub- 
stances to  the  toxin  or  bacterium  as  the  case  may  be, — these 
being  generally  spoken  of  as  anti- substances.  It  will  accordingly 
be  convenient  to  speak  of  anti-substances  in  general. 

The  development  of  anti-substances,  first  observed  in  the  case 
of  the  injection  of  toxins,  is  found  to  occur  when  a  great  many 
different  substances  are  introduced  into  the  tissues  of  the  living 
body.  We  can,  in  fact,  divide  organic  molecules  into  two  classes 
— those  which  give  rise  to  the  production  of  anti-substances,  and 
those  which  have  not  this  property.  Amongst  the  former  are 
30 


466 


IMMUNITY 


various  toxins,  ferments,  molecules  of  tissue  cells,  bacteria,  red 
corpuscles,  etc.  They  are  all  probably  of  proteid  nature,  though 
their  true  constitution  is  not  known,  and  none  of  them  have 
been  obtained  in  a  pure  condition.  Amongst  the  latter  may 
be  placed  the  various  poisons  of  known  constitution,  glucosides, 
alkaloids,  etc.  We  may  also  state  at  present  that  the  anti- 
substance  forms  a  chemical  or  physical  union  with  the  particular 
substance  which  has  led  to  its  development,  and  shall  discuss 
the  evidence  for  this  later.  Furthermore,  the  anti-substance  has 
apparently  a  specific  combining  group  which  fits,  as  it  were,  a 
group  in  the  corresponding  substance,  the  two  groups  having 
been  compared  to  a  lock  and  key.  It  is,  however,  to  be  noted 
that  this  specificity  is  a  chemical  one  rather  than  a  biological 
one.  An  anti-serum,  for  example,  developed  by  the  injection  of 
bacterium  A  may  also  have  some  effect  on  bacterium  B,  and 
thus  appear  not  to  be  specific.  We  have,  however,  evidence  to 
show  that  some  of  the  preponderating  molecules  in  bacterium  A 
are  also  present  in  bacterium  B,  and  thus  the  theory  of  chemical 
specificity  is  not  invalidated.  The  number  of  different  anti- 
substances,  as  judged  by  their  combining  properties,  would 
appear  to  be  almost  unlimited,  a  fact  which  throws  new  light 
on  the  complexity  of  the  structure  of  living  matter.  When  anti- 
substances  are  studied  as  regards  their  action  on  the  substances 
with  which  they  combine,  they  may  be  conveniently  arranged 
in  three  classes  corresponding  to  Ehrlich's  three  classes  of 
receptors  (vide  p.  491).  In  the  first  place,  the  anti-substance 
may  simply  combine  with  the  substance  without,  so  far  as  we 
know,  producing  any  change  in  it,  and  to  this  group  the  anti- 
toxins and  anti-ferments  belong.  In  the  second  place,  the 
anti-substance,  in  addition  to  combining,  may  produce  some 
recognisable  physical  alteration.  In  other  words,  it  possesses 
an  active  or  zymotoxic  group  as  well  as  a  combining  group. 
The  agglutiniris  may  be  mentioned  as  examples  of  this  group. 
In  the  third  place,  the  anti-substance  after  combination  leads  to 
the  combination  of  another  body  normally  present  in  serum 
called  complement  or  alexine,  and  this  latter,  which  has  a  con- 
stitution very  similar  to  that  of  a  toxin,  may  lead  to  physical 
change,  for  example,  death  or  solution  of  a  cell.  Anti-substances 
of  this  class  are  known  as  immune -bodies  or  amboceptors 
(Ehrlich)  or  as  sensitising  substances — substances  sensibilisatrices 
of  French  writers.  Anti-substances  of  the  second  and  third 
groups  are  met  with  especially,  though  not  exclusively,  when 
formed  elements  such  as  bacteria,  red  corpuscles,  or  tissue  cells, 
etc.,  are  injected,  the  anti-serum  developed  possessing  agglu- 


ANTITOXIC   SERUM  467 

tinating,  solvent,  or  other  properties  towards  the  particular 
substance. 

After  this  preliminary  statement  in  explanation  we  shall 
consider  the  actual  properties  of  the  two  classes  of  serum,  and 
later  we  shall  resume  the  theoretical  consideration. 

Antitoxic  Serum. — The  best  examples  are  the  antitoxic 
sera  of  diphtheria  and  tetanus,  though  similar  principles  and 
methods  are  involved  in  the  case  of  the  anti-sera  to  ricin  and 
abrin,  and  to  snake  poison.  We  shall  here  speak  of  diphtheria 
and  tetanus.  The  steps  in  the  process  of  preparation  may  be 
said  to  be  the  following :  First,  the  preparation  of  a  powerful 
toxin.  Second,  the  estimation  of  the  power  of  the  toxin.  Third, 
the  development  of  antitoxin  in  the  blood  of  a  suitable  animal 
by  gradually  increasing  doses  of  the  toxin.  Fourth,  the  estima- 
tion from  time  to  time  of  the  antitoxic  power  of  the  serum  of 
the  animal  thus  treated. 

1.  Preparation  of  the  Toxin. — The  mode  of  preparation  and 
the  conditions  affecting  the   development   of  diphtheria  toxin 
have  already  been  described  (p.  362).     In  the  case  of  tetanus  the 
growth  takes  place  in  glucose  bouillon  under  an  atmosphere  of 
hydrogen  (vide  p.   60).     In  either  case  the  culture  is  filtered 
through  a  Chamberland  filter  when  the  maximum    degree   of 
toxicity  has  been  reached.     The  term  "  toxin  "  is  usually  applied 
for  convenience  to  the  filtered  (i.e.  bacterium-free)  culture. 

2.  Estimation  of  the    Toxin. — The  power    of  the    toxin    is 
estimated  by  the  subcutaneous  injection  of  varying  amounts  in 
a  number  of  guinea-pigs,  and   the  minimum  dose  which  will 
produce   death   is   thus  obtained.      This,    of   course,    varies   in 
proportion  to  the  weight  of  the  animal,  and  is  expressed  accord- 
ingly.    In  the  case   of  diphtheria,   in  Ehrlich's  standard,   the 
minimum  lethal  dose— known  as  M.L.D. — is  the  smallest  amount 
which  will  certainly  cause  death  in  a  guinea-pig  of  250  grms. 
within  four  days.     Behring  uses  the  term  "  normal  diphtheria 
toxin  of  simple    strength"  (DTN1),    as   indicating  a   toxin    of 
which  '01  c.c.  is  the  minimum  lethal  dose  under  these  conditions. 
A  toxin  of  which  the  minimum  lethal  dose  is  '02  will  be  of  half 
normal  strength  (DTN'5) ;  and  so  on.     The  testing  of  a  toxin 
directly  is  a  tedious  process,  and  in  actual  practice,  where  many 
toxins  have  to  be  dealt  with,  it  is  found  more  convenient  to  test 
them   by  finding   how  much  will  be  neutralised  by  a  certain 
amount  of  a  standard  antitoxic  serum,  viz.  an  "immunity  unit" 
(p.  468). 

3.  Development  of  Antitoxin. — The   earlier   experiments   on 
tetanus  and  diphtheria  were  performed  on  small  animals,  such 


468  IMMUNITY 

as  guinea-pigs,  but  afterwards  the  sheep  and  the  goat  were 
used,  and  finally  horses.  In  the  case  of  the  small  animals  it  was 
found  advisable  to  use  in  the  first  stages  of  the  process  either  a 
weak  toxin  or  a  powerful  toxin  modified  by  certain  methods. 
Such  methods  are  the  addition  to  the  toxin  of  terchloride  of 
iodine  (Behring  and  Kitasato),  the  addition  of  Gram's  iodine 
solution  in  the  proportion  of  one  to  three  (Roux  and  Vaillard), 
and  the  plan,  adopted  by  Vaillard  in  the  case  of  tetanus,  of 
using  a  series  of  toxins  weakened  to  varying  degrees  by  being 
exposed  to  different  temperatures,  viz.  60°,  and  55°,  and  50°  C. 
In  the  case  of  large  animals  immunisation  is  sometimes  started 
with  small  doses  of  unaltered  toxin  ;  and  the  doses  are  gradu- 
ally increased.  The  toxin  is  at  first  injected  into  the  sub- 
cutaneous tissues,  later  into  a  vein.  Ultimately  300  c.c.,  or 
more,  of  active  diphtheria  toxin  thus  injected  may  be  borne  by 
a  horse,  such  a  degree  of  resistance  being  developed  after  the 
treatment  has  been  carried  out  for  two  or  three  months.  In  all 
cases  of  immunising  the  general  health  of  the  animal  ought  not 
to  suffer.  If  the  process  is  pushed  too  rapidly  the  antitoxic 
power  of  the  serum  may  diminish  instead  of  increasing,  and  a 
condition  of  marasmus  may  set  in  and  may  even  lead  to  the 
death  of  the  animal  (v.  p.  494).  (In  immunisation  of  small 
animals  an  indication  of  their  general  condition  may  be  obtained 
by  weighing  them  from  time  to  time.) 

4.  Estimating  the  Antitoxic  Power  of,  or  "standardising,"  the 
Serum. — This  is  done  by  testing  the  effect  of  various  quantities 
of  the  serum  of  the  immunised  animal  against  a  certain  amount 
of  toxin.  Various  standards  have  been  used,  of  which  the  two 
chief  are  that  of  Ehrlich  and  that  of  Roux.  Ehrlich  has 
adopted  as  the  immunity  unit  the  amount  of  antitoxic  serum 
which  will  neutralise  100  times  the  minimum  lethal  dose  of 
toxin,  serum  and  toxin  being  mixed  together,  diluted  up  to 
4  c.c.  and  injected  subcutaneously  into  a  guinea-pig  of  250 
grms.  weight,  the  prevention  of  the  death  of  the  animal  within 
four  days  being  taken  as  the  indication  of  neutralisation,  As  a 
standard  in  testing,  Ehrlich  employs  quantities  of  serum  of 
known  antitoxic  power  in  a  dry  condition,  preserved  in  a  vacuum 
in  a  cool  place,  and  in  the  absence  of  light.  A  thoroughly  dry 
condition  is  ensured  by  having  the  glass  bulb  containing  the 
dried  serum  connected  with  another  bulb  containing  anhydrous 
phosphoric  acid.  With  such  a  standard  test-serum  any  newly 
prepared  serum  can  readily  be  compared.  A  "  normal "  antitoxic 
serum  is  one  of  which  1  c.c.  contains  an  immunity  unit.  1  c.c. 
of  a  serum,  of  which  '02  c.c.  will  protect  from  a  hundred  times 


USE   OF   ANTITOXIC   SERA  469 

the  lethal  dose,  will  possess  50  immunity  units,  and  20  c.c.  of 
this  serum  1000  immunity  units.  Sera  have  been  prepared  of 
which  1  c.c.  has  the  value  of  800  units  or  even  more. 

Roux  adopts  a  standard  which  represents  the  animal  weight  in 
grammes  protected  by  1  c.c.  of  serum  against  the  dose  of  virulent  bacilli 
lethal  to  a  control  guinea-pig  in  thirty  hours,  the  serum  being  injected 
twelve  hours  previously.  Thus,  if  '01  c.c.  of  a  serum  will  protect  a 
guinea-pig  of  500  grins,  against  the  lethal  dose,  1  c.c.  (1  grm.)  will  protect 
50,000  grms.  of  guinea-pig,  and  the  value  of  the  serum  will  be  50,000. 

During  the  process  of  development  of  antitoxin  a  small 
quantity  of  the  blood  of  the  animal  is  withdrawn  from  time  to 
time,  and  the  antitoxic  power  tested  in  the  manner  described 
above.  After  a  sufficiently  high  degree  of  antitoxic  power  has 
been  reached  the  animal  is  bled  under  aseptic  precautions,  and 
the  serum  is  allowed  to  separate  in  the  usual  manner.  It  is  then 
ready  for  use,  but  some  weak  antiseptic,  such  as  -5  per  cent 
carbolic  acid,  is  usually  added  to  prevent  its  decomposing.  Other 
antitoxic  sera  are  prepared  in  a  corresponding  manner.  Some 
further  facts  about  antitetanic  serum  are  given  on  p.  384. 

Use  of  Antitoxic  Sera. — In  all  cases  the  antitoxic  serum  ought 
to  be  injected  as  early  in  the  disease  as  possible,  and  in  large 
doses.  In  the  case  of  diphtheria  1500  immunity  units  of  anti- 
toxic serum  was  the  amount  first  recommended  for  the  treatment 
of  a  bad  case,  but  the  advisability  of  using  larger  doses  has 
gradually  become  more  and  more  evident.  Sidney  Martin 
recommends  that  as  much  as  4000  uaits  should  be  administered 
at  once,  and  that  if  necessary  this  quantity  should  be  repeated. 
A  strong  serum  prepared  by  Behring  contains  3000  units  in 
5-6  c.c.,  but  even  stronger  sera  may  be  obtained.  Even  very 
large  doses  of  antitoxic  serum  are  without  any  harmful  effects 
beyond  the  occasional  production  of  urticarial  and  erythematous 
rashes.  Where  large  quantities  of  serum  require  to  be  ad- 
ministered, as  is  always  the  case  with  antitetanic  serum,  injections 
must  be  made  at  different  parts  of  the  body;  preferably  not 
more  than  20  c.c.  should  be  injected  at  one  place.  The  immunity 
conferred  by  injection  of  antitoxic  serum  lasts  a  comparatively 
short  time,  usually  a  few  weeks  at  longest. 

Sera  of  Animals  immunised  against  Vegetable  and  Animal 
Poisons. — It  was  found  by  Ehrlich  in  the  case  of  the  vegetable 
toxins,  ricin  and  abrin,  and  also  by  Calmette  and  Fraser  in  the 
case  of  the  snake  poisons,  that  the  serum  of  animals  immunised 
against  these  respective  substances  had  a  protective  effect  when 
injected  along  with  them  into  other  animals.  Ehrlich  found, 
for  example,  that  the  serum  of  a  mouse  which  had  been  highly 


470  IMMUNITY 

immunised  against  ricin  by  feeding  as  described  above,  could 
protect  another  mouse  against  forty  times  the  fatal  dose  of  that 
substance.  He  considered  that  in  the  case  of  the  two  poisons, 
antagonistic  substances — "anti-ricin"  and  "anti-abrin" — were 
developed  in  the  blood  of  the  highly-immunised  animals.  A 
corresponding  antagonistic  body,  to  which  Fraser  has  given  the 
name  "antivenin,"  appears  in  the  blood  of  animals  in  the  process 
of  immunisation  against  snake  poison. 

These  investigations  are  specially  instructive,  as  such  vegetable 
and  animal  poisons,  both  as  regards  their  local  action  and  the 
general  toxic  phenomena  produced  by  them,  present,  as  we  have 
seen,  an  analogy  to  various  toxins  of  bacteria. 

Nature  of  Antitoxic  Action. — This  subject  is  only  part  of  the 
general  question  with  regard  to  the  relation  of  anti-substances 
to  their  corresponding  substances,  but  it  is  with  regard  to  anti- 
toxic action  that  most  of  the  work  has  been  done.  We  have  to 
consider  here  two  points,  viz.  (a)  the  relation  of  antitoxin  to 
toxin,  and  (6)  the  source  of  the  antitoxin.  With  regard  to  the 
former  subject  there  has  been  much  diversity  of  opinion,  but 
the  evidence  now  available  goes  to  show  that  the  antagonism 
between  toxin  and  antitoxin  is  not  a  physiological  one  but  that 
the  two  bodies  unite  in  vitro  to  form  a  compound  inert  towards 
the  living  tissues,  there  being  in  the  toxin  molecule  an  atom 
group  which  has  a  specific  affinity  for  the  antitoxin  molecule  or 
part  of  it.  We  shall  consider  the  facts  in  favour  of  this  view, 
and  in  doing  so  we  must  also  take  into  account  the  anti-sera  of 
the  vegetable  toxins,  of  snake  poisons,  etc. 

When  toxin  and  antitoxin  are  brought  together  in  vitro  it 
can  be  proved  that  their  behaviour  towards  each  other  resembles 
what  is  observed  in  chemical  union.  Thus  it  has  been  found 
that  a  definite  period  of  time  elapses  before  the  neutralisation 
of  the  toxin  is  complete,  that  neutralisation  takes  place  more 
rapidly  in  strong  solutions  than  in  weak,  and  that  it  is  hastened 
by  warmth  and  delayed  by  cold.  C.  J.  Martin  and  Cherry,  and 
also  Brodie,  have  shown,  that  in  the  case  of  diphtheria  toxin  and 
in  that  of  an  Australian  snake  poison  the  toxin  molecules  will 
pass  through  a  colloid  membrane  (p.  166),  whilst  those  of  the 
corresponding  antitoxin  will  not.  Now  if  a  mixture  of 
equivalent  parts  of  toxin  and  antitoxin  is  freshly  prepared  and 
at  once  filtered,  a  certain  amount  of  toxin  will  pass  through,  but 
the  longer  such  a  mixture  is  allowed  to  stand  before  filtration 
the  less  toxin  passes,  till  a  time  is  reached  when  no  toxin  is 
found  in  the  filtrate.  Further,  if  the  portion  of  fluid  which  at 
this  stage  has  not  passed  through  the  filter  be  injected  into  an 


NATURE   OF   ANTITOXIC   ACTION  471 

animal  no  symptoms  take  place ;  this  shows  that  after  a  time 
neutralisation  is  complete.  Again,  in  cases  when  the  toxin  has 
some  definite  physical  effect  demonstrable  in  vitro,  e.g.  lysis, 
agglutination,  coagulation,  or  the  prevention  of  coagulation,  its 
action  can  be  annulled  by  the  antitoxin ;  in  such  circumstances 
manifestly  no  physiological  action  of  antitoxin  through  the 
medium  of  the  cells  of  the  body  can  come  into  play.  These 
facts  are  practically  conclusive  in  favour  of  antitoxin  action 
depending  upon  a  direct  union  of  the  two  substances  concerned. 

The  evidence  usually  brought  forward  against  the  direct  union  of 
toxin  and  antitoxin  rests  chiefly  on  certain  observations  of  Calmette, 
who  found  that  the  antitoxin  to  a  snake  venom  was  more  easily  destroyed 
by  heat  than  the  toxin,  and  stated  that  when  a  neutral  mixture  of  the 
two  was  heated  at  a  temperature  sufficient  to  destroy  free  antivenin,  the 
toxic  properties  in  part  returned.  Hence  he  concluded  that  the  two 
bodies  existed  in  an  uncombined  condition  in  the  mixture.  Martin  and 
Cherry,  however,  on  repeating  these  experiments,  found  that  the  above 
result  was  not  obtained  if  sufficient  time  for  complete  combination  was 
allowed  ;  but  if  this  precaution  was  not  taken,  then  the  presence  of  the 
free  toxin  was  revealed  when  the  antitoxin  was  destroyed  by  heat. 
Even,  however,  if  Calmette's  results  were  quite  correct,  they  cannot  be 
considered  to  constitute  a  proof  that  chemical  union  does  not  occur  : 
they  would  only  prove  that  the  toxin  has  not  been  destroyed.  If 
two  complicated  chemical  compounds  of  unequal  stability  are  in  loose 
chemical  union,  it  is  quite  conceivable  that  the  less  stable  may  be 
destroyed  (e.g.  by  heat),  whilst  the  more  stable  escapes. 

Although  practically  all  authorities  are  now  agreed  as  to  the 
direct  combination  of  toxin  and  antitoxin  there  is  still  much  uncer- 
tainty as  to  the  exact  nature  of  this  union.  Controversy  on  this 
subject  may  be  said  to  date  from  the  important  work  of  Ehrlich 
on  the  neutralisation  of  diphtheria  toxin.  Using  an  immunity 
unit  of  antitoxin  (the  equivalent  of  100  doses  of  toxin)  he  deter- 
mined with  any  example  of  crude  toxin  the  largest  amount  of 
toxin  which  could  be  neutralised  completely,  so  that  no  symptoms 
resulted  from  -an  injection  of  the  mixture.  This  amount  he 
called  the  limes  null  dose,  expressed  as  L0.  He  then  investigated 
the  effects  of  adding  larger  amounts  of  toxin  to  the  immunity 
unit  and  observed  the  quantity  which  was  first  sufficient  to 
produce  a  fatal  result,  that  is,  which  contained  one  M.L.D.  of 
free  toxin ;  this  amount  he  called  the  limes  todtlich,  fatal  limit, 
expressed  as  Lt.  Now  if,  as  he  supposed,  the  union  of  toxin  and 
antitoxin  resembled  that  of  a  strong  acid  and  base,  L^  —  L0  ought 
to  be  the  equivalent  of  a  minimum  lethal  dose  of  the  toxin  alone. 
This,  however,  was  never  found  to  be  the  case,  the  difference 
being  always  considerably  more  than  one  M.L.D.  For  example, 
in  the  case  of  one  toxin,  M.L.D.  =  '0165  gr.,  Lt.  =  1'26  gr.,  L0  = 


472  IMMUNITY 

'9  gr. ;  difference  =  '36  gr.,  i.e.  21*9  M.L.D.  This,  in  brief,  is 
what  is  known  as  the  "  Ehrlich  phenomenon,"  and  it  has  been 
explained  by  him  as  the  result  of  the  presence  of  toxoids  (vide 
p.  171),  i.e.  toxin  molecules  in  which  the  toxophorous  group  has 
become  degenerated.  He  distinguishes  three  possible  varieties 
of  such  bodies  according  to  the  affinity  of  the  haptophorous 
group,  namely  prototoxoid  with  more  powerful  affinity  than  the 
toxin  molecule,  epitoxoid  with  less  powerful  affinity,  and  syntoxoid 
with  equal  affinity.  The  presence  of  epitoxoids  would  manifestly 
explain  the  above  phenomenon.  The  L0  dose  would  represent 
toxin  +  epitoxoid  molecules  all  united  to  antitoxin  molecules  and 
the  addition  of  another  M.L.D.  of  toxin  would  not  result  in 
there  being  a  free  fatal  dose,  but  in  the  added  toxin  taking  the 
place  of  epitoxoid.  Several  lethal  doses  would  need  to  be  added 
before  the  mixture  was  sufficient  to  produce  a  fatal  result ;  that  is, 
Lt  -  L0  would  equal  several  M.L.D.s.  Ehrlich  observed  another 
fact  strongly  in  favour  of  the  existence  of  toxoids,  namely  that 
in  the  course  of  time  the  toxin  might  become  much  weakened, 
so  that  in  one  case  observed  the  M.L.D.  was  three  times  the 
original  fatal  dose,  and  still  the  amount  of  antitoxin  necessary  to 
neutralise  it  completely  was  the  same  as  before.  Ehrlich  also 
investigated  the  effects  of  partial  neutralisation  of  the  L0  amount 
of  toxin,  that  is,  he  added  to  this  amount  different  fractions  of  an 
immunity  unit  and  estimated  the  toxicity  of  the  mixture.  He 
found  by  this  method  that  the  neutralisation  of  the  toxin  did 
not  take  place  gradually,  but  as  if  there  were  distinct  bodies 
present  with  different  combining  affinities — the  graphic  repre- 
sentation of  the  mixture  not  being  a  curve  but  a  step-stair  line. 
Thus  he  distinguished  proto-,  deutero-,  and  trito-toxins  (with 
corresponding  toxoids).  It  will  thus  be  seen  that  Ehrlich  regards 
the  combination  toxin-antitoxin  to  be  a  firm  one,  and  that  the 
neutralisation  phenomena  are  to  be  explained  by  the  complicated 
constitution  of  the  crude  toxin. 

The  chief  criticism  of  Ehrlich's  views  has  come  from  the 
important  work  of  Madsen  and  Arrhenius.  Their  main  con- 
tention is  that  the  toxin-antitoxin  combination  is  not  a  firm  one 
but  a  reversible  one,  and  is  governed  by  the  laws  of  physical 
chemistry.  For  example,  in  the  case  of  a  mixture  of  ammonia 
and  boracic  acid  in  solution,  there  is  a  constant  relation  between 
the  amounts  of  each  of  the  substances  in  the  free  condition  and 
the  amounts  in  combination, — the  combination  is  reversible,  so 
that  if  some  of  the  free  ammonia  were  removed  a  certain  amount 
of  the  combined  ammonia  would  become  dissociated  to  take  its 
place ;  further,  if  to  the  mixture,  in  a  state  of  equilibrium, 


MODE   OF   PRODUCTION   OF   ANTITOXINS     473 

more  ammonia  or  more  boracic  acid  were  added,  part  would 
remain  free  while  part  would  combine.  Accordingly,  if  toxin 
and  antitoxin  behaved  in  a  similar  manner  an  explanation  of  the 
Ehrlich  phenomenon  would  be  afforded.  Madsen  and  Arrhenius 
have  worked  out  the  question  in  the  case  of  a  great  many  toxins 
and  find  that  the  graphic  representation  of  neutralisation  is  in 
every  case  a  curve  which  can  be  represented  by  a  formula.  It 
should  be  noted  in  connection  with  this  controversy  that  there 
are  two  questions  which  may  be  independent  of  each  other,  viz. 
(1)  does  the  "toxin"  in  any  particular  case  represent  a  single 
substance  or  several  1  (2)  What  is  the  nature  of  the  combination 
of  any  one  constituent  substance  and  its  anti-substance — is  it 
reversible  or  is  it  not  1  It  may  be  said  that  it  is  practically 
impossible  to  explain  the  facts  with  regard  to  diphtheria  toxin 
on  the  hypothesis  of  a  single  substance  even  if  this  should  have 
its  combining  and  toxic  actions  equally  weakened ;  "  toxoids  "  in 
Ehrlich's  sense  must  in  our  opinion  be  supposed.  Then  there  is 
an  important  fact  established  by  Danysz  and  by  v.  Dungern, 
namely  that  the  amount  of  toxin  neutralisable  by  a  given  amount 
of  antitoxin  is  different  according  as  the  toxin  is  added  in  several 
moieties  or  all  at  once — in  the  latter  case  the  amount  of  toxin 
neutralisable  is  greater.  There  seems  no  explanation  of  this 
according  to  the  view  of  Madsen  and  Arrhenius  as  the  same  state 
of  equilibrium  ought  to  be  reached  in  the  two  cases,  that  is,  the 
amounts  of  toxin  neutralised  should  be  the  same.  On  the  other 
hand  we  have  instances  of  the  combination  of  a  substance  and  its 
anti-substance  being  reversible — the  example  of  a  hsemolytic 
immune-body  may  be  cited  (p.  481) — and  there  is  no  doubt  that 
there  are  varying  degrees  of  firmness  of  the  union.  It  is  quite 
evident  that  if  there  should  be  several  toxic  bodies  in  a  "toxin," 
and  that  if  the  union  of  some  of  these  with  antitoxin  should  be 
reversible,  the  problem  becomes  one  of  extreme  complexity. 

There  has  recently  been  a  tendency  on  the  part  of  some 
authorities  to  consider  that  the  union  of  toxin-antitoxin  does  not 
correspond  to  what  takes  place  in  ordinary  chemical  union,  but 
is  a  physical  interaction  of  bodies  in  a  colloidal  state,  the  action 
being  one  of  the  so-called  absorption  phenomena.  The  smaller 
toxin  molecule  becomes  entangled,  as  it  were,  in  the  larger 
antitoxin  one,  very  much  as  a  dye  becomes  attached  to  the 
structure  of  a  thread.  Bordet  has  long  maintained  a  theory  of 
this  nature  and  gives  reasons  for  believing  that  there  is  no 
definite  quantitative  relationship  in  the  combination  of  the 
molecules  of  the  two  substances,  different  amounts  of  antitoxin 
affecting  in  varying  degree  all  the  molecules  of  a  given  amount 


474  IMMUNITY 

of  toxin.  A  statement  on  the  general  question  is  at  present 
impossible ;  we  can  only  say  that  combination  of  the  two  bodies 
does  occur ;  that  sometimes,  probably  often,  the  "  toxin  "  con- 
tains different  toxic  bodies  with  varying  affinity ;  and  that  in  a 
few  instances  the  combination  has  been  proved  to  be  reversible, 
but  to  what  extent  this  is  the  case  remains  still  to  be  determined. 
The  next  question  to  be  considered  is  the  source  of  antitoxin. 
The  following  three  possibilities  present  themselves  :  (a)  antitoxin 
may  be  formed  from  the  toxin,  i.e.  may  be  a  "  modified  toxin  "  ; 
(6)  antitoxin  may  be  the  result  of  an  increased  formation  of 
molecules  normally  present  in  the  tissues ;  (c)  antitoxin  may  be 
an  entirely  new  product  of  the  cells  of  the  body.  It  can  now  be 
stated  that  antitoxin  is  not  a  modified  toxin.  It  has  been  shown, 
for  example,  that  the  amount  of  antitoxin  produced  by  an  animal 
may  be  many  times  greater  than  the  equivalent  of  toxin  injected  ; 
and  further,  that  when  an  animal  is  bled  the  total  amount  of 
antitoxin  in  the  blood  may  some  time  afterwards  be  greater 
than  it  was  immediately  after  the  bleeding,  even  although  no 
additional  toxin  is  introduced.  This  latter  circumstance 
shows  that  antitoxin  is  formed  by  the  cells  of  the  body.  If 
antitoxin  is  a  product  of  the  cells  of  the  body,  we  are  almost  com- 
pelled, on  theoretical  grounds,  to  conclude  that  it  is  not  a  newly- 
manufactured  substance,  but  a  normal  constituent  of  the  living 
cells  which  is  produced  in  increased  quantity.  We  have,  however, 
direct  evidence  of  the  presence  of  antitoxin  under  normal  con- 
ditions,— the  presence  of  such  being  shown  by  its  uniting  with 
toxin  and  rendering  it  inert.  Normal  horse  serum,  to  mention 
an  example,  may  have  a  varying  amount  of  antitoxic  action  to 
the  diphtheria  poison,  ox-bile  has  a  similar  action  to  snake  poison, 
whilst  in  the  case  of  other  anti-substances — such  as  agglutinins, 
bacteriolysins,  hsemolysins,  etc. — whose  production  is  governed 
by  the  same  laws,  numerous  examples  might  be  given.  It  is, 
however,  rather  to  the  protoplasm  of  living  cells  than  to  the  serum 
that  we  must  look  for  the  source  of  antitoxins.  In  the  first  place, 
we  have  evidence  that  in  the  living  body  bacterial  toxins  enter 
into  combination  with,  or,  as  it  is  often  expressed,  are  fixed  by 
the  tissues — presumably  by  means  of  certain  combining  affinities. 
This  has  been  shown  by  the  experiments  of  Donitz  and  of 
Heymans  with  tetanus  toxin.  We  have,  in  such  cases,  however, 
no  evidence  as  to  where  the  toxin  is  fixed  beyond  that  supplied 
by  the  occurrence  of  symptoms.  Another  line  of  research  which 
has  been  followed  is  to  bring  emulsions  of  various  organs  into 
contact  with  a  given  toxin  and  observe  whether  any  of  the 
toxicity  is  removed.  This  was  first  carried  out  by  Wassermann 


CHEMICAL   NATURE   OF   ANTITOXINS        475 

and  Takaki,  who  investigated  the  action  of  emulsions  of  the 
central  nervous  system  of  the  susceptible  guinea-pig  on  tetanus 
toxin.  They  found  in  this  way  that  the  nervous  system  con- 
tained bodies  which  had  a  neutralising  effect  on  the  toxin. 
For  example,  it  was  shown  that  1  c.c.  of  emulsion  of  brain  and 
spinal  cord  was  capable  of  protecting  a  mouse  against  ten  times 
the  fatal  dose  of  toxin.  These  observations  have  been  confirmed, 
though  their  significance  has  been  variously  interpreted.  It 
would,  however,  be  out  of  place  to  discuss  at  length  the  opposing 
views,  and  we  accordingly  simply  state  the  facts  ascertained. 
We  may  note,  however,  that  it  is  not  a  serious  objection  that  in 
certain  animals  other  tissues  than  that  of  the  central  nervous 
system  can  combine  with  tetanus  toxin — this  might  take  place 
with  or  without  resulting  symptoms  :  the  important  fact  is  that 
in  the  nervous  system  certain  molecules  have  an  affinity  for  the 
toxin. 

It  will  be  seen  from  what  has  been  stated  with  regard  to  the 
relation  of  toxin  and  antitoxin,  that  the  fixation  of  toxin  by  the 
tissues  leads  up  theoretically  to  the  possible  production  of  anti- 
toxin. In  other  words,  the  substance  which,  when  forming  part 
of  the  cells,  fixes  the  toxin  and  thus  serves  as  the  means  of 
poisoning,  may  act  as  an  antitoxin  when  free  in  the  blood. 
This  will  be  discussed  below  in  connection  with  Ehrlich's  theory 
of  passive  immunity.  We  may  conclude  by  saying  that  anti- 
toxin is  probably  represented  by  molecules  normally  present  in 
the  cells  or  (more  rarely)  in  the  fluids  of  the  body. 

Of  the  chemical  nature  of  antitoxins  we,  know  little.  From 
their  experiments  C.  J.  Martin  and  Cherry  deduce  that  while 
toxins  are  probably  of  the  nature  of  albumoses,  the  antitoxins 
probably  have  a  molecule  of  greater  size,  and  may  be  allied  to 
the  globulins.  Hiss  and  Atkinson  have  also  come  to  the  con- 
clusion that  antitoxin  belongs  to  the  globulins.  They  found 
that  the  precipitate  with  magnesium  sulphate  from  anti- 
diphtheria  serum  contained  practically  all  the  antitoxin,  and  that 
any  substance  obtained  which  had  an  antitoxic  value  gave  all 
the  reactions  of  a  globulin.  They  also  found  that  the  per- 
centage amount  of  globulin  precipitated  from  the  serum  of  the 
horse  increased  after  it  was  treated  in  the  usual  way  for  the 
production  of  antitoxin.  Such  a  supposed  difference  in  the 
sizes  of  the  molecules  might  explain  the  fact,  observed  by 
Fraser  and  also  by  C.  J.  Martin,  that  antitoxin  is  much  more 
slowly  absorbed  when  introduced  subcutaneously  than  is  the  case 
with  toxin. 

Antitoxin,  when  present  in  the  serum,  leaves  the  body  by 


476  IMMUNITY 

the  various  secretions,  and  in  these  it  has  been  found,  though 
in  much  less  concentration  than  in  the  blood.  It  is  present  in 
the  milk,  and  a  certain  degree  of  immunity  can  be  conferred  on 
animals. by  feeding  them  with  such  milk,  as  has  been  shown  by 
Ehrlich,  Klemperer,  and  others.  Klemperer  also  found  traces  of 
antitoxin  in  the  yolk  of  eggs  of  hens  whose  serum  contained 
antitoxin.  Bulloch  also  found  in  the  case  of  hsemolytic  sera 
(vide  infra)  that  the  anti-substance  ("  immune  -body  ")  is  trans- 
mitted from  the  mother  to  the  offspring. 

Antibacterial  Serum. — The  stages  in  the  preparation  of 
antibacterial  sera  correspond  to  those  in  the  case  of  antitoxic 
sera,  but  living,  or,  in  the  early  stages,  dead  cultures  are  used 
instead  of  toxin  separated  by  nitration,  and  in  order  to 
obtain  a  serum  of  high  antibacterial  power  a  very  virulent 
culture  in  large  doses  must  be  ultimately  tolerated  by  the 
animal.  For  this  purpose  a  fairly  virulent  culture  is  obtained 
fresh  from  a  case  of  the  particular  disease,  and  its  virulence 
may  be  further  increased  by  the  method  of  passage.  This 
method  of  obtaining  a  high  degree  of  immunity  against  the 
microbe  is  specially  applicable  in  the  case  of  those  organisms 
which  invade  the  tissues  and  multiply  to  a  great  extent  within 
the  body,  and  of  which  the  toxic  effects,  though  always  existent, 
are  proportionately  small  in  relation  to  the  number  of  organisms 
present.  The  method  has  been  applied  in  the  case  of  the 
typhoid  and  cholera  organisms,  the  bacillus  of  bubonic  plague, 
the  bacillus  coli  communis,  the  pneumococcus,  streptococcus 
(Marmorek),  and  many  others.  In  fact,  it  seems  capable  of  very 
general  application. 

The  important  result  obtained  by  such  experiments  is,  that  if 
an  animal  be  highly  immunised  by  the  method  mentioned,  the 
development  of  the  immunity  is  accompanied  by  the  appearance 
in  the  blood  of  protective  substances,  which  can  be  transferred  to 
another  animal.  The  law  enunciated  by  Behring  regarding 
immunity  against  toxins  thus  holds  good  in  the  case  of  the 
living  organisms,  as  was  first  shown  by  PfeifFer.  The  latter 
found,  for  example,  that  in  the  case  of  the  cholera  organism,  so 
high  a  degree  of  immunity  could  be  produced  in  the  guinea-pig, 
that  "002  c.c.  of  its  serum  would  protect  another  guinea-pig 
against  ten  times  the  lethal  dose  of  the  organisms,  when  in- 
jected along  with  them.  Here  again  is  presented  the  remark- 
able potency  of  the  antagonising  substances  in  the  serum, 
which  in  this  case  lead  to  the  destruction  of  the  corresponding 
microbe. 

The  anti-streptococcic  serum  of  Marmorek  may  be  briefly  described,  as 


PROPERTIES   OF  ANTIBACTERIAL   SERUM     477 

it  has  come  into  extensive  practical  use.  This  observer  found  that  he 
could  intensify  the  virulence  of  a  streptococcus  by  growing  it  alternately 
in  the  peritoneal  cavity  of  a  guinea-pig  and  in  a  mixture  of  human  blood 
serum  and  bouillon  (vide  p.  41).  The  virulence  became  so  enormously 
increased  by  this  method,  that  when  only  one  or  two  organisms  were 
introduced  into  the  tissues  of  a  rabbit  a  rapidly  fatal  septicaemia  was 
produced.  Streptococci  of  this  high  degree  of  virulence  were  used  first 
by  subcutaneous,  afterwards  by  intravenous  injection,  to  develop  a  high 
degree  of  resistance  in  the  horse.  Injections  were  continued  over  a  con- 
siderable period  of  time,  and  the  protective  power  of  the  serum  was 
tested  by  mixing  it  with  a  certain  dose  of  the  virulent  organisms,  and 
then  injecting  into  a  rabbit.  The  serum  of  a  horse  highly  immunised  in 
this  way  constitutes  the  anti-streptococcic  serum  which  has  been 
extensively  used  with  success  in  many  cases  of  streptococcic  invasion  in 
the  human  subject.  Marmorek,  however,  found  that  this  serum  had 
little  antitoxic  power,  that  is,  could  only  protect  from  a  comparatively 
small  dose  of  toxin  obtained  by  filtration  of  cultures. 

Anti-typhoid,  anti-cholera,1  anti-pneumococcic,  anti-plague, 
and  other  sera  are  all  prepared  in  an  analogous  manner. 

Properties  of  Antibacterial  Serum.  —  We  have  here  to 
consider  the  three  main  actions  mentioned  above,  viz.  (a) 
bactericidal  and  lysogenic  action,  (6)  opsonic  action,  and  (c) 
agglutinative  action. 

(a)  Bactericidal  and  Lysogenic  Action. — Pfeiffer  found  that 
if  certain  organisms,  e.g.  the  cholera  spirillum,  were  injected 
into  the  peritoneal  cavity  of  a  guinea-pig  highly  immunised 
against  these  organisms  they  lost  their  motility  almost  immedi- 
ately, gradually  became  granular,  swollen,  and  then  disappeared 
in  the  fluid — these  changes  constitute  "  PfeifFer's  phenomenon." 
Further,  he  found  that  the  same  phenomenon  was  witnessed  if 
a  minute  quantity  of  the  anti-serum  was  added  to  a  certain 
quantity  of  the  corresponding  organisms,  and  the  mixture 
injected  into  the  peritoneal  cavity  of  a  non-treated  animal. 
Pfeiffer  found  that  the  serum  of  convalescent  cholera  patients 
gave  the  same  reaction  as  that  of  immunised  animals.  He 
obtained  the  same  reaction  also  in  the  case  of  the  typhoid 
bacillus  and  other  organisms.  From  his  observations  he  con- 
cluded that  the  reaction  was  specific,  and  could  be  used  as  a 
means  of  distinguishing  organisms  which  resemble  one  another. 
He  accordingly  considered  that  a  specific  substance  was  developed 
in  the  process  of  immunisation  and  that  this  was  rendered 
actively  bactericidal  by  the  aid  of  the  living  cells  of  the  body. 
It  was  subsequently  shown,  however,  by  Metchnikoff  and  by 
Bordet  that  lysogenesis  might  occur  outside  the  body  by  the 

1  A  true  antitoxic  cholera  serum  has  been  prepared  by  Metchnikoff,  E. 
Roux,  and  Taurelli-Salimbeni. 


478  IMMUNITY 

addition  of  fresh  peritoneal  fluid  or  normal  serum  to  the  heated 
immune-serum.  Pfeiffer  also  found  that  an  anti-serum  heated  to 
70°  C.  for  an  hour  produced  the  reaction  when  injected  with  the 
corresponding  organisms  into  the  peritoneum  of  a  fresh  animal. 
The  outcome  of  these  and  subsequent  researches  is  to  show  that 
when  an  animal  is  immunised  against  a  bacterium  a  substance 
appears  in  its  serum  with  combining  affinity  for  that  particular 
organism.  This  substance  which  is  generally  known  as  the 
immune-body,  amboceptor  (Ehrlich),  or  substance  sensibilisatrice 
(Bordet)  is  comparatively  stable,  resisting  usually  a  temperature 
of  70°  C  for  an  hour.  It  cannot  produce  the  destructive  effect 
alone  but  requires  the  addition  of  a  substance  normally  present 
in  the  serum,  which  is  spoken  of  under  various  names — com- 
plement (Ehrlich),  alexine  or  cytase  (French  writers).  The  com- 
plement is  relatively  unstable,  being  rapidly  destroyed  by  a 
temperature  of  60°  C.,  and  it  is  not  increased  in  amount  during 
the  process  of  immunisation.  Though  ferment-like  in  its  in- 
stability, it  differs  from  a  ferment  in  being  fixed  or  used  up  in 
definite  quantities. 

The  phenomenon  of  lysogenesis  is,  however,  only  seen  in  the 
case  of  certain  organisms  when  an  animal  is  highly  immunised 
against  them ;  the  typhoid  and  cholera  group  are  outstanding 
examples.  It  is  also  to  be  noted  that  it  sometimes  is  seen  in  the 
case  of  a  normal  serum  (vide  Natural  Immunity).  In  other  cases 
the  bactericidal  effect  of  a  serum  may  occur  without  the  rapid  dis- 
solution characteristic  of  lysogenesis  though  other  structural 
changes  may  be  produced.  In  still  other  cases  a  bactericidal 
effect  may  be  wanting ;  nevertheless  it  may  be  shown  that  an 
immune-body  is  developed  by  the  process  of  immunisation.  This 
may  be  done  by  observing  the  increased  amount  of  complement 
which  is  fixed  through  the  medium  of  the  anti-serum  (immune- 
body),  sensitised  red  corpuscles  being  used  as  the  test  for  the 
presence  of  free  complement.  The  following  scheme  will  show 
the  mode  of  experiment,  which  is  carried  out  in  a  series  of 
small  test-tubes  : — 

(1)  Bacteria  +  immune-body  (anti-serum  heatedat  55°  C.)  +  complement. 
(The  same  amount  of  bacteria  and  immune-body  in  each  tube,  varying 
amounts  of  complement  in  different  tubes.) 

(2)  Incubate  at  37°  C.  for  one  and  a  half  hours. 

(3)  Add  to  each  tube  red  corpuscles  treated  with  the  corresponding 
immune-body,  and  incubate  for  another  hour. 

The  "  immune-body  "  is  in  each  case  the  anti-serum  deprived 
of  complement  (by  heating  at  55°  C.),  obtained  from  animals 
injected  with  the  bacteria  and  red  corpuscles  respectively.  The 


H^EMOLYTIC   AND   OTHER   SERA  479 

control  is  got  by  substituting  in  another  experiment  the  same 
amount  of  heated  normal  serum  for  the  anti-serum.  If  there  is 
free  complement  left  there  will  be  corresponding  lysis  of  the  red 
corpuscles  ;  if  the  complement  has  all  been  fixed  there  will  be  no 
lysis.  To  take  an  example  from  Muir's  experiments, — it  was  found 
that  an  emulsion  of  the  bacteria  alone  took  up  '03  c.c.  of  guinea- 
pig's  complement,  whilst  the  same  amount  of  bacteria  treated 
with  immune-body  took  up  '13  c.c.  The  all-important  action  of 
the  immune-body  is  thus  to  bring  an  increased  amount  of  com- 
plement into  union  with  bacteria  ;  whether  death  of  the  bacteria 
will  result  or  not  will  depend  ultimately  on  their  sensitiveness 
to  the  action  of  the  particular  complement. 

It  is  to  be  noted  that  with  a  bactericidal  serum  there  is 
an  optimum  amount  of  immune-body  which  gives  the  greatest 
bactericidal  effect.  If  this  amount  be  exceeded  the  bactericidal 
action  becomes  diminished  and  may  be  practically  annulled. 
This  result,  which  is  generally  known  as  the  Neisser-Wechsberg 
phenomenon,  has  been  the  subject  of  much  controversy,  and 
cannot  yet  be  said  to  be  satisfactorily  explained.  It  would 
accordingly  be  out  of  place  to  discuss  here  the  different  views 
with  regard  to  it.  (Regarding  some  theoretical  considerations 
as  to  the  therapeutic  applications  of  antibacterial  sera,  vide 
p.  489.) 

The  laws  of  lysogenesis  are,  however,  not  peculiar  to  the 
case  of  solution  of  bacteria  by  the  fluids  of  the  body,  but,  as  has 
been  shown  within  the  last  few  years,  hold  also  in  the  case  of 
other  organised  substances,  red  corpuscles,  leucocytes,  etc.,  when 
these  are  introduced  into  the  tissues  of  an  animal  as  in  a  process 
of  immunisation.  Of  such  sera  the  hsemolytic  have  been  most 
fully  studied,  and  owing  to  the  delicacy  of  the  reaction  and  the 
ease  with  which  it  can  be  observed,  have  been  the  means  of 
throwing  much  light  on  the  process  of  lysogenesis,  and  thus  on 
one  part  of  the  subject  of  immunity.  A  short  account  of  their 
properties  may  now  be  given. 

Hcemolytic  and  other  Sera. — It  has  been  known  for  some  time 
that  in  some  instances  the  blood  serum  of  one  animal  has,  in 
certain  degree,  the  power  of  dissolving  the  red  corpuscles  of 
another  animal  of  different  species ;  in  other  instances,  how- 
ever, this  property  cannot  be  detected.  Bordet  showed  that 
if  one  animal  were  treated  with  repeated  injections  of  the 
corpuscles  of  another  of  different  species,  the  serum  of  the  former 
acquired  a  marked  hsemolytic  property  towards  the  corpuscles  of 
the  latter,  the  property  being  demonstrated  when  the  serum  is 
added  to  the  corpuscles.  Bordet  also  found  that  the  haemolytic 


480  IMMUNITY 

property  disappeared  when  the  haemolytic  serum  was  heated  at 
55°  C.,  but,  as  in  the  case  of  a  bacteriolytic  serum,  was  regained 
on  the  subsequent  addition  of  some  serum  from  a  fresh  (i.e.  non- 
treated)  animal.  These  observations  have  been  fully  confirmed 
and  greatly  extended.  Ehrlich  and  Morgenroth  analysed  the 
phenomena  in  question,  and  showed  that  the  specially  developed 
and  heat-resisting  substance,  "immune-body,"  entered  into  com- 
bination with  the  red  corpuscles  at  a  comparatively  low 
temperature,  namely  at  0°  C. ;  whereas  complement  does  not 
combine  at  this  temperature.  In  this  way  a  method  is  supplied 
by  which  the  immune-body  can  be  removed  from  a  hsemolytic 
serum  while  the  complement  is  left.  They  came  to  the  con- 
clusion that  immune  -  body  combined  with  the  complement 
though  the  combination  was  less  firm  and  only  occurred 
at  a  higher  temperature — best  about  37°  C.  They  therefore 
consider  that  the  immune-body  acts  as  a  sort  of  connecting  link 
between  the  red  corpuscle  and  the  complement,  hence  the  term 
"amboceptor"  which  Ehrlich  afterwards  applied.  It  may  be 
stated,  however,  that  the  direct  union  of  complement  and 
immune-body  has  not  been  conclusively  demonstrated.  Bordet, 
on  the  other  hand,  holds  that  the  immune-body  acts  merely  as 
a  sensitising  agent — hence  the  term  substance  sensibilisatrice — 
and  allows  the  ferment-like  complement  to  act.  It  is  quite 
evident  from  his  writings,  however,  that  he  does  not  mean,  as 
is  often  assumed,  that  the  immune-body  causes  some  lesion  in 
the  corpuscle  which  allows  the  complement  to  act,  but  simply 
that  it  produces  in  the  molecules  (receptors)  of  the  red  corpuscles 
an  avidity  for  complement.  All  that  we  can  say  definitely  at 
present  is  that  the  combination  of  receptor  +  immune-body  takes 
up  complement  in  firm  union  while  neither  does  so  alone ; 
whether  the  immune-body  acts  as  a  link  between  the  two  or  not 
must  be  left  an  open  question.  Even  after  the  corpuscles  are 
laked  with  water  the  receptors  are  not  destroyed:  Muir  and 
Ferguson  have  shown  that  they  can  still  take  up  immune- 
body  and,  through  its  medium,  complement,  just  as  the  intact 
corpuscles  do.  Ehrlich  and  Morgenroth  showed  that  in  some 
cases  the  red  corpuscles  can  take  up  much  more  immune-body 
than  is  necessary  for  their  lysis,  and  Muir  found  in  one  case 
studied,  that  each  further  dose  of  immune-body  led  to  the  fixation 
of  more  complement,  so  that  as  many  as  ten  times  the  hsemolytic 
dose  of  complement  might  thus  be  used  up.  It  is  a  matter  of 
considerable  importance  that  the  union  of  immune-body  and  red 
corpuscles  can  be  shown  to  be  a  reversible  action.  If,  as  was 
found  by  Morgenroth  and  Muir  independently,  corpuscles  treated 


H^EMOLYTIC   AND   OTHER   SERA  481 

with  several  doses  of  immune-body  and  then  repeatedly  washed 
in  salt  solution  be  mixed  with  untreated  corpuscles  and  allowed 
to  remain  for  an  hour,  then  sufficient  immune-body  will  pass 
from  the  former  to  the  latter,  so  that  all  become  lysed  on 
the  addition  of  sufficient  complement.  The  combination  of 
complement,  on  the  other  hand,  is  usually  of  very  firm  nature. 
It  has  been  a  disputed  point  whether  there  are  several  distinct 
complements  in  a  normal  serum  with  different  relations  to 
different  immune-bodies,  for  which  Ehrlich  and  his  co-workers 
have  brought  forward  a  large  amount  of  evidence,  or  whether, 
as  Bordet  holds,  there  is  a  single  complement  which  may,  how- 
ever, show  slight  variations  in  behaviour  towards  different 
immune-bodies.  There  is  at  least  no  doubt  that  all  the  com- 
plement molecules  in  a  serum  are  not  the  same.  Workers  of 
the  French  school  also  hold  that  complement  does  not  exist 
in  the  free  condition  in  the  blood,  but  is  liberated  from  the 
leucocytes  when  the  blood  is  shed ;  though  this  cannot  be  held 
as  proved,  there  is  evidence  that  the  amount  of  free  complement 
increases  after  the  blood  is  shed  and  some  time  later  gradually 
diminishes. 

The  haemolytic  action  of  a  normal  serum  can  be  shown  in 
many  cases  to  be  of  the  same  nature  as  that  of  an  immune- 
serum,  that  is,  complement  and  the  homologue  of  an  immune- 
body  can  be  distinguished.  For  example,  the  guinea-pig's  serum 
is  haemolytic  to  the  ox's  corpuscles ;  if  a  portion  of  serum  be 
heated  at  55°  C.  the  complement  will  be  destroyed ;  if  another 
portion  be  treated  with  ox's  corpuscles  at*0°  C.,  the  natural 
immune-body  will  be  removed  and  only  complement  will  be  left. 
Neither  portion  is  in  itself  hsemolytic,  but  this  property  becomes 
manifest  again  when  the  two  portions  are  mixed.  Haemolytic 
sera  are  of  great  service  in  the  study  of  the  question  of  specificity. 
Each  is  specific  in  the  sense  already  explained  (p.  466),  but  the 
serum  developed  against  the  corpuscles  of  an  animal  may  have 
some  action  on  those  of  an  allied  species,  that  is,  some  receptors 
are  common  to  the  two  species.  This  fact  can  be  readily  shown 
by  the  usual  absorption  tests,  for  example,  in  the  case  of  an 
anti-ox  serum  tested  on  sheep's  corpuscles.  A  close  analogy 
holds  to  what  has  been  established  in  the  case  of  agglutinins. 
It  is  further  of  great  interest  to  note  that  by  the  injection  of  red 
corpuscles  into  an  animal  its  serum  not  only  becomes  haemolytic, 
but  in  many  cases  when  heated  at  55°  C.  possesses  also  agglu- 
tinating and  opsonic  properties  towards  the  red  corpuscles  used. 
And  further,  it  would  appear  that  in  some  cases  at  least  the 
immune-body,  hsemagglutinin,  and  haemopsonin  are  distinct 
31 


482  IMMUNITY 

substances.  These  facts  abundantly  show  how  close  an  analogy 
obtains  between  anti-bacterial  and  hsemolytic  sera,  and  how 
important  a  bearing  hsemolytic  studies  have  on  the  questions  of 
immunity  in  general. 

In  addition  to  hsemolytic  sera,  anti-sera  have  been  obtained 
by  the  injection  of  leucocytes,  spermatozoa,  ciliated  epithelium, 
liver  cells,  nervous  tissue,  etc.  The  laws  governing  the  pro- 
duction and  properties  of  these  are  identical,  that  is,  each  serum 
exhibits  a  specific  property  towards  the  body  used  in  its  produc- 
tion— i.e.  dissolves  leucocytes,  immobilises  spermatozoa,  etc. 
The  specificity  is,  however,  not  so  marked  as  in  the  case  of 
sera  produced  against  red  blood  corpuscles ;  thus  a  serum  pro- 
duced against  tissue  cells  is  often  haemolytic ;  this  is  probably 
due  to  various  cells  of  the  body  having  the  same  receptors. 
Here  again  when  the  anti-serum  produces  no  destructive  effect 
on  the  corresponding  cells,  the  presence  of  an  immune-body  may 
be  demonstrated  by  the  increased  amount  of  complement  which 
is  taken  up  through  its  medium.  It  may  also  be  mentioned 
that  each  anti-serum  usually  exhibits  toxic  properties  towards 
the  animal  whose  cells  have  been  used  in  the  injections,  e.g.  a 
hsemolytic  serum  may  produce  a  fatal  result,  with  signs  of 
extensive  blood  destruction,  hsemoglobinuria,  etc.,  i.e.  it  is 
hsemotoxic  for  the  particular  animal ;  a  serum  prepared  by 
injection  of  liver  cells  has  been  found  to  produce  on  injection 
necrotic  changes  in  the  liver  in  the  species  of  animal  whose  liver 
cells  were  used.  These  are  mentioned  as  examples  of  a  very 
large  group  of  specific  activities. 

With  regard  to  the  sites  of  origin  of  immune-bodies  our 
information  is  still  very  deficient.  Pfeiffer  and  Marx  brought 
forward  evidence  in  the  case  of  typhoid,  and  Wassermann  in  the 
case  of  cholera,  that  the  immune-bodies  are  chiefly  formed  in 
the  spleen,  lymphatic  glands,  and  bone-marrow.  According  to 
certain  workers  of  the  French  school,  the  chief  source  of  anti- 
substances  acting  on  cells  such  as  red  blood  corpuscles  is  the  large 
mononucleated  leucocytes,  whilst  those  acting  on  bacteria  are 
chiefly  derived  from  the  polymorpho-nuclear  leucocytes  (vide 
p.  495).  Another  view  is  that  immune-bodies  are  chiefly  formed 
by  the  large  mononucleated  leucocytes,  whilst  complements  are 
products  of  the  polymorphs.  That  these  cells  are  concerned  in 
the  production  of  antagonistic  and  protective  substances  is 
almost  certain,  though  another  possible  source  of  wide  extent, 
viz.  the  endothelium  of  the  vascular  system,  has  been  largely 
overlooked.  As  yet,  definite  statements  cannot  be  made  on  this 
point. 


OPSONIC   ACTION  483 

Methods  of  Hsemolytic  Tests. — A  hsemolytic  serum  is  usually  pre- 
pared by  injecting  the  red  corpuscles  of  an  animal  into  the  peritoneum 
of  an  animal  of  different  species — the  corpuscles  of  the  ox  are  most 
frequently  used,  and  the  rabbit  is  the  most  suitable  animal  for  injection. 
The  corpuscles  ought  to  be  completely  freed  of  serum  by  repeatedly 
washing  them  in  sterile  salt  solution  and  centrifugalising.  An  injection 
of  the  corpuscles  of  5  c.c.  of  ox's  blood  followed  by  two  injections,  each 
of  10  c.c.,  at  intervals  of  ten  days,  will  usually  give  an  active  serum. 
The  animal  should  be  killed  by  bleeding  it,  aseptically  as  far  as  possible, 
seven  to  ten  days  after  the  last  injection  ;  the  serum  which  separates 
may  be  collected  in  suitable  lengths  of  quill  glass-tubing  drawn  out  at 
the  ends,  which  are  afterwards  sealed  in  the  flame.  To  ensure  sterility 
when  the  serum  is  to  be  kept  some  time,  it  is  advisable  to  heat  it  for  an 
hour  at  55°  C.  on  three  successive  days  ;  we  have  always  found  that 
serum  treated  in  this  way  remains  sterile.  It  is  of  course  devoid  of 
complement.  The  test  amount  of  corpuscles  is  usually  1  c.c.  of  a  5  per 
cent  suspension  of  corpuscles  in  '8  per  cent  sodium  chloride  solution  : 
that  is,  the  corpuscles  of  5  c.c.  blood  are  completely  freed  of  serum  by 
repeatedly  washing  in  salt  solution,  and  then  salt  solution  is  added  to 
make  up  100  c.c.  In  any  investigation  it  is  necessary  to  obtain  the 
minimum  hsemolytic  dose  (M.H.D.)  of  the  immune-body  and  of  the 
complement  to  be  used.  (It  is  to  be  noted  that  as  complement  does  not 
increase  during  immunisation,  the  hsemolytic  dose  of  the  fresh  serum 
will  come  far  short  of  representing  the  amount  of  immune-body  present.) 
In  testing  the  dose  of  immune-body,  the  fresh  serum  to  be  used  as  com- 
plement must  be  devoid  of  haemolytic  action  (in  the  present  instance 
rabbit's  serum  will  be  found  suitable)  and  more  than  sufficient  to  produce 
lysis  with  immune-body  is  added  to  each  of  a  series  of  tubes.  Varying 
amounts  of  immune-body  are  added  to  the  tubes,  the  contents  are 
shaken,  made  up  to  1'5  c.c.  and  incubated  for  two  hours.  The  amount 
of  lysis  is  then  noted  and  the  tubes  are  placed  in  a  cool  chamber  till 
next  morning,  when  a  final  reading  is  taken.  The  smallest  amount  of 
immune-body  which  gives  complete  lysis  is  of,, course  the  M.H.D.  : 
sometimes  this  may  be  as  low  as  *001  c.c.  for  the  test  amount  of 
corpuscles.  When  further  observations  are  to  be  continued  on  the  same 
day,  the  reading  after  incubation  must  be  taken  as  the  working 
standard.  To  estimate  the  M.H.D.  of  complement,  proceed  in  a 
corresponding  manner  ;  to  each  of  a  series  of  tubes  add  several  doses  of 
immune-body,  and  then  to  the  several  tubes  different  amounts  of  com- 
plement. The  activity  of  a  serum  as  complement  varies  considerably, 
and  each  sample  must  be  separately  tested.  The  above  will  serve  as  an 
indication  of  the  fundamental  methods  ;  for  further  details  special 
papers  on  the  subject  must  be  consulted. 

(b)  Opsonic  Action. — The  presence  of  a  substance  in  an 
immune-serum  which  makes  the  corresponding  organism  sensitive 
to  phagocytosis  was  first  demonstrated  by  Denys  and  Leclef  in 
1895,  in  the  case  of  an  anti-streptococcal  serum.  They  also 
showed  that  the  serum  produced  this  effect  by  acting  on  the 
organism,  not  on  the  leucocytes.  It  is,  however,  chiefly  to  the 
researches  of  Wright  and  his  co-workers  that  this  subject  has 
come  into  special  prominence.  Wright  and  Douglas  in  their 
first  paper  showed  that  the  phagocytosis  of  staphylococci  by 


484  IMMUNITY 

leucocytes  depended  on  a  body  in  the  normal  serum  which 
became  fixed  to  the  cocci  and  made  them  a  prey  to  the 
phagocytes.  To  this  they  gave  the  name  of  "  opsonin  "  (vide 
pp.  194,  261).  There  is  no  phagocytosis  of  cocci  by  leucocytes 
washed  in  salt  solution ;  normal  serum  heated  to  55°  C.  is  also 
without  effect  in  inducing  this  phenomenon.  They  could  not 
demonstrate  any  effect  of  the  opsonin  on  the  leucocytes.  On  the 
other  hand,  if  bacteria  be  exposed  to  the  fresh  serum,  and  they 
be  freed  from  the  excess  of  serum  and  then  exposed  to  phago- 
cytes also  washed  free  from  serum,  they  will  be  readily  taken 
up  by  the  cells.  It  has  been  abundantly  shown  that  the  opsonic 
action  of  the  serum  is  increased  by  the  process  of  immunisation 
against  an  organism,  and  the  opsonic  index  represents  the  degree 
of  immunity  in  one  of  its  aspects  as  already  explained  (p.  Ill ). 
The  matter  has,  however,  become  complicated  by  the  circum- 
stance that  in  an  immune-serum  an  opsonin  may  still  be  present 
after  the  serum  is  heated  at  55°  C.,  as  has  been  shown  by  Dean 
and  others.  Some  observers  consider  that  this  opsonin  is  simply 
an  immune-body,  but  the  results  brought  forward  by  others  would 
point  to  their  being  different  substances,  at  least  in  certain  cases, 
notably  in  hsemolytic  sera.  We  are,  however,  probably  safe  in 
saying  that  the  thermostable  opsonin  of  an  immune-serum  is  a 
true  anti-substance,  possessing  the  specific  characters  of  anti- 
substances  in  general  and  comparable  in  this  respect  and  in  its 
mode  of  production  with  an  agglutinin.  Muir  and  Martin  have, 
however,  found  that  the  thermolabile  opsonin  of  a  normal  serum 
has  different  characters.  For  example,  when  a  normal  serum  is 
tested  on  a  particular  bacterium,  the  opsonic  effect  on  that 
bacterium  may  be  removed  by  treating  the  serum  with  other 
bacteria ;  in  other  words,  the  thermolabile  opsonin  of  normal 
serum  does  not  possess  the  specific  character  of  the  opsonin 
developed  in  the  process  of  immunisation.  They  have  also 
found  that  various  substances  or  combinations  of  substances 
which  act  as  "complement -absorbers"  also  remove  the  opsonic 
property  from  a  normal  serum,  while  they  have  no  effect  on  an 
immune-opsonin.  According  to  this  view  the  opsonic  effect  of 
the  unheated  serum  of  an  actively  immunised  animal  or  person 
would  represent  the  sum  of  the  effects  of  the  two  kinds  of 
opsonin. 

Further  study  will  be  necessary  before  the  exact  relationships 
of  these  substances  are  fully  understood,  and  other  questions  with 
regard  to  them  have  as  yet  scarcely  been  touched  upon. 
Increased  phagocytic  action  had  long  been  known  by  the  work 
of  Metchnikoff  to  be  associated  with  the  development  of  active 


AGGLUTINATION  485 

immunity  and  the  theory  of  stimulation  of  leucocytes  was 
supported  by  many.  The  work  on  opsonins  has  caused  a  swing 
of  the  pendulum  in  the  other  direction,  and  points  to  the 
development  of  anti-substances  in  the  serum  as  the  all-important 
factor.  It  remains  to  be  determined  to  what  extent  the  opsonic 
and  directly  bactericidal  properties  taken  together  will  explain 
the  phenomena  of  natural  and  acquired  immunity. 

(c)  Agglutination. — Charrin  and  Roger  in  1889  observed 
that  when  the  bacillus  pyocyaneus  was  grown  in  the  serum  of 
an  animal  immunised  against  this  organism,  the  growth  formed 
a  deposit  at  the  foot  of  the  vessel ;  whereas  a  growth  in  normal 
serum  produced  a  uniform  turbidity.  Gruber  and  Durham,  in 
investigating  Pfeiffer's  reaction,  found  that  when  a  small  quantity 
of  an  anti-serum  is  added  to  an  emulsion  of  the  corresponding 
bacterium,  the  organisms  become  agglutinated  into  clumps, 
this  phenomenon  depending  upon  the  presence  of  definite  bodies 
in  the  serum  called  agglutinins. 

It  had  been  already  found  that  the  serum  of  convalescents 
from  typhoid  fever  could  protect  animals  to  a  certain  extent 
against  typhoid  fever,  and,  in  view  of  the  facts  experimentally 
established,  it  appeared  a  natural  proceeding  to  enquire  whether 
such  serum  possessed  an  agglutinative  action  and  at  what  stage 
of  the  disease  it  appeared.  The  result,  obtained  independ- 
ently by  Griinbaum  and  Widal,  but  first  published  by  the  latter, 
was  to  show  that  the  serum  possessed  this  specific  action  shortly 
after  infection  had  taken  place ;  in  other  words,  the  develop- 
ment of  this  variety  of  anti- substance  can  fee  demonstrated  at 
an  early  stage  of  the  disease.  Agglutination  is  also  observed  in 
the  case  of  cholera,  Malta  fever,  bacterial  dysentery,  glanders, 
plague,  infection  by  Gartner's  bacillus,  b.  coli,  etc.  Furthermore 
the  phenomenon  is  not  peculiar  to  bacteria ;  it  is  seen,  for 
example,  when  an  animal  is  injected  with  the  red  corpuscles  of 
another  species,  haemagglutinins  appearing  in  the  serum,  which 
have  a  corresponding  specificity. 

The  physical  changes  on  which  agglutination  depends  cannot 
as  yet  be  said  to  be  fully  understood.  Gruber  and  Durham 
considered  that  the  agglutinin  produced  a  change  in  the  envelope 
of  the  bacterium,  causing  it  to  swell  up  and  become  viscous,  and 
the  facts  first  established  appeared  to  be  in  favour  of  this  view. 
On  the  other  hand,  this  is  not  the  full  explanation,  as  it  has 
been  shown  by  Nicolle  and  by  Kruse  that  if  an  old  bacterial 
culture  be  filtered  through  porcelain,  the  addition  of  some  of 
the  corresponding  anti-serum  produces  a  sort  of  granular 
precipitate  in  it,  and  that  when,  as  in  the  agglutination  of  bacteria, 


486  IMMUNITY 

minute  inorganic  particles  are  added  to  the  mixture  they  become 
aggregated  into  clumps.  The  phenomenon  would  thus  appear 
to  be  the  result  of  the  interaction  of  the  agglutinin  and  some 
substance  in  the  bacterial  cell  which  is  known  as  the  agglutin- 
able  substance  or  as  the  agglutinogen,  seeing  that  it  is  probably 
the  element  in  the  bacterial  structure  which  in  the  tissues  of  the 
animal  or  person  leads  to  the  development  of  the  agglutinin. 
Joos  has  found  in  the  case  of  the  typhoid  bacillus  that 
there  are  two  agglutinable  substances  which  differ  in  their 
resistance  to  heat — a-  and  /^-agglutinogen,  and  that  they  give  rise 
to  corresponding  agglutinins.  Further,  as  the  result  of  a  com- 
parative study  of  the  agglutinins  of  a  motile  and  a  non-motile 
variety  of  the  hog  cholera  bacillus  Theobald  Smith  has  come 
to  the  conclusion  that  there  is  an  agglutinin  which  is  produced 
by  and  acts  on  the  fiagella  and  another  which  is  similarly  related 
to  the  bacterial  bodies.  The  former  acts  in  very  much  higher 
dilutions  than  the  latter,  and  this  is  regarded  as  an  explanation 
of  the  fact  that  in  the  case  of  non-motile  organisms  the 
agglutinating  serum  acts  only  in  proportionately  high  concentra- 
tion as  compared  with  the  case  of  most  motile  forms.  Another 
factor  necessary  for  the  phenomenon  of  agglutination  is  a  proper 
salt  content.  Bordet  showed  that  if  the  clumps  of  agglutinated 
bacteria  are  freed  from  salt  by  washing  in  distilled  water  they 
become  resolved,  and  that  on  the  addition  of  some  sodium 
chloride  they  are  formed  again,  and  Joos  has  also  brought 
forward  striking  confirmatory  evidence  as  to  the  necessity  for 
the  presence  of  salts.  It  is  thus  probable  that  in  the  pheno- 
menon of  agglutination  as  ordinarily  understood  more  than  one 
factor  is  concerned,  and  it  is  possible  that  in  part  it  may  depend 
on  some  altered  molecular  relationship  of  the  bacteria  to  the 
surrounding  fluid  analogous  to  altered  surface  tension. 

As  stated  above,  the  agglutinins  are  usually  placed  in  the 
second  order  of  anti-substances,  and  are  regarded  as  possessing 
a  combining  group  and  an  active  or  agglutinating  group.  The 
constitution  would  thus  be  analogous  to  that  of  a  toxin,  and  in 
conformity  with  this  view  Eisenberg  and  Volk  consider  that  the 
agglutinating  group  may  be  destroyed  while  the  combining 
group  remains,  the  result  being  an  agglutinoid.  The  evidence 
for  this  lies  in  the  fact  that  when  an  agglutinating  serum  is 
heated  to  a  certain  temperature,  not  only  does  it  lose  its 
agglutinating  action  but  when  the  bacteria  are  treated  with 
such  a  serum  their  agglutination  by  active  serum  is  interfered 
with,  a  sort  of  plugging  up  of  the  combining  molecules  having 
apparently  taken  place.  Other  facts  have,  however,  been 


AGGLUTINATION  487 

brought  forward  in  opposition  to  this  view,  and  the  existence  of 
agglutinoids  cannot  be  said  to  be  completely  proved.  Like 
immune-bodies  agglutinins  are  not  destroyed  at  55°  C.  (a 
temperature  sufficient  to  annul  bactericidal  action),  and  the 
question  arises  as  to  the  relation  of  the  two  bodies ;  discussion 
has  also  taken  place  as  to  how  far  agglutination  is  an  indication 
of  immunity.  It  may  be  said  that  in  the  case  of  certain  sera 
investigated  it  has  been  shown  that  the  immune-body  and  the 
agglutinin  are  separate  substances,  and  that  the  agglutinative 
power  does  not  vary  pari  passu  with  the  degree  of  immunity — 
a  serum  may  be  strongly  agglutinative  and  feebly  bactericidal 
and  vice  versa.  But  while  probably  as  a  rule  the  two  substances 
are  distinct,  it  would  not  be  justifiable  to  say  this  is  always  the 
case — that  is,  that  an  immune-body  never  has  an  agglutinating 
action.  And  while  the  agglutinative  power  cannot  in  itself  be 
taken  as  the  measure  of  the  degree  of  immunity,  agglutinins  and 
immune-bodies  are  the  products  of  corresponding  reactive  pro- 
cesses, and  their  formation  is  governed  by  corresponding  laws. 
Agglutinins  become  fixed  in  definite  proportion  by  the  receptors 
of  the  bacteria  ;  that  is,  the  agglutinin  becomes  used  up  in  the 
process  of  agglutination,  and  it  has  been  shown  that  bacteria 
may  take  up  many  times  the  amount  necessary  to  their 
agglutination — a  corresponding  fact  to  what  has  been  established 
with  regard  to  immune -bodies  of  ha3molytic  sera.  The 
agglutinins  are  specific  in  the  sense  which  has  been  explained 
above  (p.  466).  It  can  be  shown  by  the  method  of  absorption 
that  in  an  agglutinating  serum  there  may  be^several  agglutinins 
with  different  combining  groups,  some  of  which  may  be  taken  up 
by  organisms  allied  to  that  which  has  given  rise  to  the  anti- 
serum.  Whether  or  not  the  combination  of  an  agglutinin  with 
the  bacterial  receptors  is  a  reversible  action  must  be  left  an  open 
question. 

Besides  those  stated  above,  other  phenomena  have  been 
observed  in  the  interaction  of  anti-sera  and  the  corresponding 
bacteria.  For  example,  it  has  been  shown  that  when  certain 
bacteria — e.g.  the  typhoid  bacillus,  b.  coli,  and  b.  proteus — are 
grown  in  bouillon  containing  a  small  proportion  of  the  homo- 
logous serum,  their  morphological  characters  may  be  altered, 
growth  taking  place  in  the  form  of  threads  or  chains  which  are 
not  observed  in  ordinary  conditions.  In  other  instances  a  serum 
may  inhibit  some  of  the  vital  functions  of  the  corresponding 
bacterium. 

Precipitins. — This  subject  does  not  strictly  belong  to  bacteriology, 
but  the  general  phenomena  are  so  closely  allied  to  those  just  described, 


488  IMMUNITY 

especially  to  agglutination,  that  some  reference  may  be  made  to  it. 
When  the  serum  of  an  animal  is  injected  in  repeated  doses  into  another 
animal  of  different  species,  after  the  type  of  an  immunisation,  there 
appears  in  the  serum  of  the  animal  treated  a  substance  called  precipitin, 
which  causes  a  cloudiness  or  precipitate  when  added  to  the  serum  used. 
This  precipitate  results  from  the  union  of  the  precipitin  in  the  anti-serum 
with  a  body  in  the  homologous  serum,  the  latter  being  known  as  the 
precipitinogen.  (In  the  case  of  rabbits  doses  of  3  to  4  c.c.  of  the  serum 
may  be  injected  intraperitoneally  at  intervals  of  four  to  five  days,  a 
precipitin  usually  appearing  at  the  end  of  about  three  weeks.)  The 
reaction,  which  is  a  very  delicate  one,  is  conveniently  observed  by  adding 
a  given  amount  of  the  anti-serum,  say  '05  c.c.,  to  varying  amounts  of 
the  homologous  serum  '1,  '01,  etc.  c.c.,  in  a  series  of  small  test  tubes, 
the  volume  being  then  made  up  with  salt  solution  to  1  c.c.  In  this  way 
a  definite  reaction  may  be  observed  with  '001  c.c.  of  the  serum  or  even 
less.  The  reaction  is  specific  in  the  sense  explained  above.  It  is  always 
most  marked  towards  the  serum  of  the  species  used  in  the  immunisation  ; 
but  while  this  is  so,  there  may  also  be  a  slight  reaction  towards  animals  of 
allied  species.  An  anti-human  serum,  for  example,  gives  the  maximum 
reaction  with  human  serum,  but  also  a  slight  reaction  with  the  serum 
of  monkeys,  especially  of  anthropoid  apes  ;  it,  however,  gives  no  reaction 
with  the  serum  of  other  animals.  The  precipitin  test  has  thus  come  to 
be  employed  as  a  means  of  differentiating  human  from  other  bloods. 
Another  interesting  phenomenon  is  what  is  known  as  the  "deviation  of 
complement,"  which  is  produced  by  the  combination  of  the  two  sub- 
stances in  the  serum  and  anti-serum  respectively.  If  mixtures  be  made 
according  to  the  above  method,  and  then  a  small  quantity  of  complement, 
say  fresh  guinea-pig  serum,  be  added,  it  will  be  found  that  the  comple- 
ment becomes  absorbed,  as  may  be  shown  by  subsequently  adding  a  test 
amount  of  sensitised  red  blood  corpuscles.  This  deviation  phenomenon 
is  even  a  more  delicate  reaction  than  the  precipitin  test,  it  being  often 
possible  to  demonstrate  by  its  use  from  a  tenth  to  a  hundredth  of  the 
smallest  amount  of  serum  which  will  give  a  perceptible  precipitate  ;  it 
also  is  specific  within  the  same  limits.1 

Therapeutic  Effects  of  Anti-Sera. — As  will  have  been 
gathered,  the  chief  human  diseases  treated  by  anti-sera  are 
diphtheria,  tetanus,  streptococcus  infection,  pneumonia,  plague, 
and  snake  bite.  Of  the  results  of  such  treatment  most  is  known 
in  the  case  of  diphtheria.  Here  a  very  great  diminution  in  the 
mortality  has  resulted.  The  diphtheria  antitoxin  came  into 
general  use  about  October  1894,  and  the  statistics  published  by 
Behring  towards  the  end  of  1895  indicated  results  which  have 
since  been  confirmed.  In  the  Berlin  Hospitals  the  average 
mortality  for  the  years  1891-93  was  36*1  per  cent,  in  1894  it 
was  21 '1  per  cent,  and  in  January-July  1895,  14*9  per  cent.  The 
objection  that  in  some  epidemics  a  very  mild  type  of  disease 
prevails  is  met  by  the  fact  that  similar  diminutions  of  mortality 

1  For  an  account  of  precipitins,  vide  Nuttall,  "  Blood  Immunity  and 
Relationships,"  Cambridge  1904  ;  and  of  complement  deviation,  Muir  and 
Martin,  Journ.  of  Hyg.  vi.,  1906,  p.  265. 


THERAPEUTIC   EFFECTS   OF   ANTI-SERA       489 

have  occurred  all  over  the  world.  Loddo  collected  the  results 
of  7000  cases  in  Europe,  America,  Australia,  and  Japan,  in 
which  the  mortality  was  20  per  cent  as  compared  with  a  former 
mortality  in  the  same  hospitals  of  44  per  cent.  It  has  also  been 
observed  that  if  during  an  epidemic  the  supply  of  serum  fails, 
the  mortality  at  once'  rises ;  and  in  two  instances  recorded  it 
was  doubled.  It  must  here  be  remembered  that  from  the 
spread  of  bacteriological  knowledge  the  diagnosis  of  diphtheria 
is  now  much  more  accurate  than  formerly.  Another  effect  of 
the  antitoxic  treatment  has  been  that  when  tracheotomy  is 
necessary  the  percentage  of  recoveries  is  now  much  higher,  being 
73  per  cent  instead  of  27  per  cent  in  a  group  of  cases  collected 
by  the  American  Pediatric  Society.  In  the  London  fever 
hospitals  since  1894  the  recoveries  after  tracheotomy  have  been 
56  4  as  compared  with  32'1  per  cent  previous  to  the  intro- 
duction of  antitoxin.  One  of  the  most  striking  results  obtained 
in  the  same  hospitals  is  a  reduction  of  the  death-rate  in  post- 
scarlatinal  diphtheria  from  50  per  cent  to  between  4  per  cent 
and  5  per  cent.  As  the  disease  here  occurs  while  the  patient  is 
under  observation  the  treatment  is  nearly  always  begun  on  the 
first  day.  It  is  a  matter  of  prime  importance  that  the  treat- 
ment should  be  commenced  whenever  the  disease  is  recognised. 
Behring  showed  that  in  cases  treated  on  the  first  and  second 
days  of  the  disease  the  mortality  was  only  7 '3  per -cent,  and  this 
has  been  generally  confirmed,  whilst  after  the  fifth  day  it  was  of 
little  service  to  apply  the  treatment.  In  order  to  obtain  such 
results  it  cannot  be  too  strongly  insisted  mi  that  attention 
should  be  given  to  the  dosage.  When  bad  results  are  obtained 
it  may  be  strongly  suspected  that  this  precaution  has  not  been 
observed.  In  the  treatment  of  acute  tetanus  by  the  antitoxin 
the  improvement  in  results  has  not  been  marked,  but  some 
chronic  cases  have  been  benefited,  and  as  already  stated  (p.  386) 
better  results  are  obtained  in  acute  cases  if  intravenous  in- 
jection be  practised.  In  the  case  of  Yersin's  anti-plague  serum, 
though  benefit  has  appeared  to  follow  its  use,  experience 
with  its  effects  has  been  too  limited  to  enable  a  judgment 
to  be  formed.  The  same  may  be  said  to  be  true  of  the  anti- 
streptococcic  and  anti-pneumonic  sera,  though  in  the  case  of  the 
first  mentioned  numerous  cases  of  apparently  successful  result 
have  been  recorded.  With  regard  to  anti-venin,  Lamb  has  shown 
that,  if  a  cobra  with  full  glands  bites  a  man,  many  times  the 
minimal  lethal  dose  are  probably  injected.  In  cases  of  slight 
bite,  however,  benefit  may  accrue  from  the  use  of  the  anti- 
serum. 


490  IMMUNITY 

As  has  been  shown  above,  antibacterial  sera  require  for  their 
complete  action  a  sufficiency  of  complements,  and  as  these 
diminish  in  amount  when  a  serum  is  kept,  the  unsatisfactory 
results  with  this  class  of  sera  may  be  due  to  a  deficiency  of 
complement.  Or  it  may  be  as  Ehrlich  has  suggested,  that  the 
complement  naturally  existing  in  human  serum  does  not  suit 
the  immune-body  in  the  anti-serum — that  is,  is  not  taken  up 
through  the  medium  of  the  latter  and  brought  into  combination 
with  the  bacterium.  And  there  is  still  the  further  possibility 
that  even  though  the  complement  should  be  taken  up,  the 
zymotoxic  group  of  the  latter  is  not  sufficiently  active  towards 
the  bacterium  to  effect  its  death.  In  both  cases  it  will  appear 
that  an  extracellular  bactericidal  action  cannot  be  produced  by 
the  particular  immune-body  in  association  with  the  complement 
of  the  animal  in  question.  There  is  no  doubt  that  this  question 
of  complements  is  one  of  importance,  and  that  both  combining 
affinity  and  toxic  action  of  complements  must  be  considered  in 
each  case. 

Theories  as  to  Acquired  Immunity. 

The  advances  made  within  recent  years  in  our  knowledge 
regarding  artificial  immunity,  and  the  methods  by  which  it  may 
be  produced,  have  demonstrated  the  insufficiency  of  various 
theories  which  had  been  propounded.  Only  a  short  reference 
need  be  made  to  these.  The  theory  oj  exhaustion,  with  which 
Pasteur's  name  is  associated,  supposed  that  in  the  body  of  the 
living  animal  there  are  substances  necessary  for  the  existence  of 
a  particular  organism,  which  become  used  up  during  the  sojourn 
of  that  organism  in  the  tissues ;  this  pabulum  being  exhausted 
the  organisms  die  out.  Such  a  supposition  is,  of  course,  quite 
disproved  by  the  facts  of  passive  immunity.  According  to  the 
theory  of  retention  the  bacteria  within  the  body  were  considered 
to  produce  substances  which  are  inimical  to  their  growth,  so  that 
they  die  out,  just  as  they  do  in  a  test-tube  culture  before  the 
medium  is  really  exhausted.  Such  a  theory  only  survives  now 
in  the  view  that  antitoxins  are  modified  toxins,  the  evidence 
against  which  has  already  been  discussed  (p.  470).  There  then 
came  the  humoral  theory  and  the  theory  of  phagocytosis,  but 
neither  of  these  is  tenable  in  its  pure  form,  and  the  distinction 
between  them  need  not  be  maintained.  For,  on  the  one  hand, 
any  substance  with  specific  property  in  the  serum  must  be  the 
product  of  cellular  activity,  and  on  the  other  hand,  the  facts 
with  regard  to  passive  immunity  go  far  beyond  the  ingestive 
and  digestive  properties  of  phagocytes,  though  these  cells  may 


EHRLICH'S   SIDE-CHAIN   THEORY  491 

be  in  part  the  source  of  important  bodies  in  the  serum.  At  the 
present  time  interest  centres  around  two  theories,  viz.  Ehrlich's 
side-chain  theory  and  Metchnikoff' s  phagocytic  theory  as  further 
developed.  These  will  now  be  discussed,  and  it  may  be  noted 
that  the  ground  covered  by  each  is  not  coextensive.  For  the 
former  deals  chiefly  with  the  production  of  anti-substances  and 
its  biological  significance,  the  latter  deals  with  the  defensive 
properties  of  cells,  either  directly  by  their  phagocytic  activity 
or  indirectly  by  substances  produced  by  them  after  the  manner 
of  digestive  ferments.  It  will  be  seen,  however,  that  each  has 
a  normal  process  as  its  basis,  viz.  that  of  nutrition. 

1.  Ehrlich's  Side-Chain  Theory. — This  may  be  said  to  be  an 
application  of  his  views  regarding  the  nourishment  of  proto- 
plasm. A  molecule  of  protoplasm  (in  the  general  sense)  may  be 
regarded  as  composed  of  a  central  atom  group  or  executive 
centre  (Leistungskern)  with  a  large  number  of  side -chains 
(Seitenketten),  i.e.  atom  groups  with  'combining  affinity  for 
food-stuffs.  It  is  by  means  of  these  latter  that  the  living 
molecule  is  increased  in  tfre  process  of  nutrition,  and  hence 
the  name  receptors  given  by  Ehrlich  is  on  the  whole  preferable. 
These  receptors  are  of  three  chief  kinds  corresponding  to  the 
classes  of  anti-substances  described  (p.  466) ;  the  first  has  a 
single  unsatisfied  combining  group  and  fixes  molecules  of 
simpler  constitution — receptor  of  the  first  order;  the  second 
has  a  combining  group  for  the  food  molecule,  and  another 
active  or  zymotoxic  group,  which  leads  to  some  physical  change 
in  it — receptor  of  the  second  order;  the  third  has  two  com- 
bining groups,  one  for  the  food  molecule  and  another  which 
fixes  a  ferment  in  the  fluid  medium  around — receptor  of  the 
third  order  or  amboceptor.  These  latter  receptors  come  into 
action  in  the  case  of  larger  food  molecules  which  require  to  be 
broken  up  by  ferment -action  for  the  purposes  of  the  cell 
economy.  In  considering  the  application  of  this  idea  to  the 
facts  of  passive  immunity,  it  must  be  kept  in  view  that  all  the 
substances  to  which  anti-substances  have  been  obtained  are,  like 
proteids,  of  unknown  but  undoubtedly  of  very  complex  chemical 
constitution,  and  that  in  apparently  every  case  the  anti-substance 
enters  into  combination  with  its  corresponding  substance.  The 
dual  constitution  of  toxins  and  kindred  substances,  as  already 
described  (p.  170),  is  also  of  importance  in  this  connection. 
Now,  to  take  the  case  of  toxins,  when  these  are  introduced  into 
the  system  they  are  fixed,  like  food-stuffs,  by  their  haptophorous 
groups  to  the  receptors  of  the  cell  protoplasm,  but  are  unsuitable 
for  assimilation.  If  they  are  in  sufficiently  large  amount  the 


492  IMMUNITY 

toxophorous  part  of  the  toxin  molecule  produces  that  disturb- 
ance of  the  protoplasm  which  is  shown  by  symptoms  of 
poisoning.  If,  however,  they  are  in  smaller  dose,  as  in  the 
early  stages  of  immunisation,  fixation  to  the  protoplasm  occurs 
in  the  same  way ;  and  as  the  combination  of  receptors  with 
toxin  is  supposed  to  be  of  firm  nature,  the  receptors  are  lost 
for  the  purposes  of  the  cell,  and  the  combination  E-.-T.  (receptor 
+  toxin)  is  shed  off  into  the  blood.  The  receptors  thus  lost 
become  replaced  by  new  ones,  and  when  additional  toxin 
molecules  are  introduced,  these  new  receptors  are  used  up  in 
the  same  manner  as  before.  As  a  result  of  this  repeated  loss 
the  regeneration  of  the  receptors  becomes  an  over-regeneration, 
and  the  receptors  formed  in  excess  appear  in  the  free  condition 
in  the  blood  stream  and  then  constitute  antitoxin  molecules. 
There  are  thus  three  factors  in  the  process,  namely,  (1)  fixation 
of  toxin,  (2)  over-production  of  receptors,  (3)  setting  free  of 
receptors  produced  in  excess.  Accordingly  these  receptors 
which,  when  forming  part  of  the  cell  protoplasm,  anchor  the 
toxin  to  the  cell,  and  thus  are  essential  to  the  occurrence  of 
toxic  phenomena,  in  the  free  condition  unite  with  the  toxin,  and 
thus  prevent  the  toxin  from  combining  with  the  cells  and  exert- 
ing a  pathogenic  action.  The  three  orders  of  receptors,  when 
separated  from  the  cells,  thus  give  the  three  kinds  of  anti- 
substances.  Ehrlich  does  not  state  what  cells  are  specially 
concerned  in  the  production  of  anti-substances,  but  from  what 
has  been  stated  it  is  manifest  that  any  cell  which  fixes  a  toxin 
molecule,  for  example,  is  potentially  a  source  of  antitoxin. 
Cells,  to  whose  disturbance,  resulting  from  the  fixation  of  toxin, 
characteristic  symptoms  of  poisoning  are  due,  will  thus  be 
sources  of  antitoxin,  e.g.  cells  of  the  nervous  system  in  the  case 
of  tetanus,  though  the  cells  not  so  seriously  affected  by  toxin 
fixation  may  act  in  the  same  way.  The  experimental  investiga- 
tion of  the  source  of  antitoxins  has,  however,  yielded  little  result, 
and  no  definite  statement  can  be  made  on  the  subject. 

When  we  come  to  consider  how  far  Ehrlich's  theory  is  in 
harmony  with  known  facts,  we  find  that  there  is  much  in  its 
favour.  In  the  first  place,  it  explains  the  difference  between 
active  and  passive  immunity,  e.g.  difference  in  duration,  etc. ;  in 
the  former  the  cells  have  acquired  the  habit  of  discharging  anti- 
substances,  in  the  latter  the  anti-substances  are  simply  present 
as  the  result  of  direct  transference.  It  is  also  in  harmony  with 
the  action  of  antitoxins,  etc.,  as  detailed  above,  and  especially  it 
affords  an  explanation  of  the  multiplicity  of  anti-substances. 
For,  if  we  take  the  case  of  antitoxins,  we  see  that  this  depends 


EHRLICH'S   SIDE-CHAIN   THEORY  493 

upon  the  combining  affinity  of  the  toxin  for  certain  of  the  cells 
of  the  body,  and  this  again  is  referred  back  to  the  complicated 
constitution  of  living  protoplasm.  Furthermore,  the  biological 
principle  involved  is  no  new  one,  being  simply  that  of  over- 
regeneration  after  loss.  It  would  appear  likely  that  the  integrity 
of  the  executive  centres  of  the  protoplasm  molecules  would  be 
essential  to  the  satisfactory  production  of  side- chains,  and  this 
would  appear  in  accordance  with  the  fact  that  antitoxin 
formation  occurs  most  satisfactorily  when  there  is  no  marked 
disturbance  of  the  health  of  the  animal. 

It  is  to  be  noted,  however,  that  it  does  not  explain  active 
immunity  apart  from  the  presence  of  anti-substances  in  the 
serum.  For  example,  an  animal  may  be  able  to  withstand  a 
much  larger  amount  of  toxin  than  could  be  neutralised  by  the 
total  amount  of  antitoxin  in  its  serum.  This  might  theoretically 
be  explained  by  supposing  a  special  looseness  of  the  cell  re- 
ceptors so  that  the  toxin-receptor  combination  became  readily  cast 
off.  The  question,  however,  arises  whether  there  may  not  be  really 
an  increased  resistance  of  the  cells  to  the  toxophorous  affinities. 
An  observation  recently  made  by  Meyer  and  Ransom  (v.  p.  383) 
is  also  difficult  of  explanation  according  to  the  view  that  antitoxin 
is  formed  by  the  cells  with  which  the  toxin  combines  and  on 
which  it  acts.  They  found  that  in  an  animal  actively  immun- 
ised against  tetanus  and  with  antitoxin  beginning  to  appear  in 
its  blood,  the  injection  of  a  single  M.L.D.  of  tetanus  toxin  into  a 
peripheral  nerve  brought  about  tetanus  with  a  fatal  result.  On 
the  other  hand,  the  injection  of  antitoxin  into  the  sciatic  nerve 
above  the  point  of  injection  of  toxin  prevented  the  latter  from 
reaching  the  cells  of  the  cord.  One  can  scarcely  imagine  an 
explanation  of  these  facts  if  antitoxin  molecules  were  in  process 
of  being  shed  off  by  the  cells  of  the  nervous  system.  Further, 
when  the  serum  of  an  animal  contains  a  large  amount  of  anti- 
toxin, how  does  the  additional  toxin  injected  reach  the  cells 
in  order  to  influence  them  as  we  know  it  does?  This  also  is 
difficult  to  understand  unless  the  toxin  has  a  greater  affinity  for 
the  receptors  in  the  cells  than  for  the  free  receptors  (antitoxin) 
in  the  serum.  A  supersensitiveness  of  the  nerve-cells  of  an 
animal  to  tetanus  toxin,  sometimes  observed  even  when  there  is 
a  large  amount  of  antitoxin  in  the  serum,  has  been  often  brought 
forward  as  an  objection.  But  this  also  may  perhaps  be  explained 
by  there  having  occurred  a  partial  damage  of  the  cell  protoplasm 
by  the  toxophorous  action  in  the  process  of  immunisation — an 
explanation  which,  of  course,  demands  that  in  some  way  the 
freshly  introduced  toxin  may  reach  the  cells  in  spite  of  the  anti- 


494  IMMUNITY 

toxin  in  the  blood.  Further  investigation  alone  will  settle  these 
and  various  other  disputed  points,  and  may  remove  many  of  the 
apparent  objections.  At  present  we  may  say,  however,  that 
Ehrlich's  theory  is  the  only  one  which  even  attempts  to  explain 
the  cardinal  facts  of  this  aspect  of  immunity. 

In  connection  with  the  condition  of  supersensitiveness  referred  to 
above,  an  interesting  phenomenon  lias  recently  been  described  by 
Theobald  Smith,  and  is  now  generally  known  as  "serum  anaphylaxis," 
or  "Theobald  Smith's  phenomenon."  It  is  briefly  the  following  : — If  a 
guinea-pig  be  injected  with  a  quantity,  say  5  c.c.,  of  horse  serum,  no 
disturbance  follows  ;  if,  however,  the  animal  be  previously  treated, 
say  fourteen  days  before,  with  a  very  small  quantity  of  horse  serum, 
'001  c.c.  (even  less  is  sufficient),  and  then  the  5  c.c.  of  serum  be  injected, 
the  animal  usually  dies  within  an  hour  with  characteristic  symptoms. 
The  general  lesions  are  of  hsemorrhagic  nature,  as  pointed  out  by  Gay  and 
Southard,1  and  occur  especially  in  the  stomach.  The  condition  of  super- 
sensitiveness  to  the  horse  serum  lasts  fora  long  period  of  time.  Accord- 
ing to  Gay  and  Southard  the  phenomenon  depends  upon  a  substance  in 
the  horse  serum  which  they  call  anaphylactin,  and  which  persists  for  a 
long  period  of  time  in  the  blood  of  the  guinea-pig.  This  body  they  con- 
sider to  act  as  a  slight  irritant  to  the  cells  of  the  guinea-pig,  and  to 
produce  an  increased  affinity  for  the  molecules  in  the  horse  serum.  Ac- 
cordingly when  the  second  injection  is  made  the  rapid  combination  of  these 
substances  with  the  cells  result  in  the  disturbances  referred  to.  What- 
ever may  be  the  explanation,  the  phenomenon  is  of  extreme  importance 
as  showing  the  profound  alterations  in  metabolism  which  may  be  induced 
by-  a  minute  quantity  of  serum  of  a  normal  animal. 

The  facts  relating  to  hypersensitiveness  raise  the  question  of 
whether  in  any  immunisation  procedure  an  injury  may  not  be 
constantly  done  to  the  cells  forming  the  anti-substances.  We 
have  already  drawn  attention  to  the  occurrence  of  what  Wright 
has  called  the  negative  phase  in  the  course  of  the  increase  of  the 
opsonic  power  of  the  serum  aimed  at  in  a  bacterial  vaccination. 
There  is  evidence  that  such  negative  phases  are  common  in  all 
immunisations.  They  have  been  also  noted  in  the  formation  of 
antitoxins,  of  immune-bodies,  and  of  agglutinins.  Thus  in  the  case 
of  the  first,  Salamonsen  and  Madsen  showed  that  the  fall  in  the 
content  of  an  animal's  serum  in  antitoxin  after  a  fresh  toxin  injec- 
tion was  greater  than  could  be  accounted  for  by  the  neutralisa- 
tion of  the  free  antitoxin  in  the  blood  by  the  toxin  introduced, 
and  they  attributed  the  occurrence  to  an  injury  to  the  producing 
cells  temporarily  diminishing  the  productive  activity.  The 
normal  course  of  every  immunisation  may  be  said  to  consist  in 
a  succession  of  positive  and  negative  phases,  and  an  effective 
immunisation  is  one  where  each  succeeding  positive  phase  brings 

1   Vide  Gay  and  Southard,  Journ.  Med.  Research,  xvi.,  1907,  143. 


THE  THEORY  OF  PHAGOCYTOSIS     495 

the  serum  to  a  higher  content  in  anti-body.  Again,  in  no  case 
is  the  capacity  of  producing  anti-bodies  unlimited.  In  certain 
reactions  the  limit  of  possible  increase  is  less  than  in  others. 
Thus  it  is  not  possible  to  raise  the  opsonic  power  of  a  serum 
higher  than  a  not  very  great  multiple  of  its  original  opsonic 
content.  On  the  other  hand,  when  we  are  dealing  with  the 
reaction  against  bacterial  toxins  we  find  that  the  mechanism 
producing  antitoxin  can  react  in  an  extraordinary  degree,  and 
a  serum  many  thousand  times  stronger  than  that  produced 
during  the  early  days  of  immunisation  may  ultimately  be 
attained.  The  animal  body  also  exhibits  great  power  of  forming 
agglutinins,  and  the  capacity  of  forming  immune  bodies  seems 
to  occupy  an  intermediate  position  between  the  opsonic  reaction 
and  the  antitoxin  reaction.  But  even  in  the  antitoxin  reaction 
a  time  comes  in  a  high  immunisation  when  evidence  of  exhaus- 
tion of  the  producing  mechanism  is  manifest,  so  that  the  injection 
of  fresh  toxin  is  no  longer  efficient,  and  the  negative  phase  is 
not  followed  by  a  positive  phase.  From  the  practical  stand- 
point it  is  the  aim  of  the  immuniser  to  select  the  time  just 
preceding  such  an  event  for  the  bleeding  of  an  animal.  If  the 
cells  of  the  latter  be  given  a  few  months  rest  then  the  capacity 
for  producing  antitoxin  usually  reappears.  But  such  facts 
emphasise  what  we  have  said  as  to  the  possibility  of  every 
immunisation  entailing  the  infliction  of  an  injury  on  some  bodily 
mechanism. 

2.  The  Theory  of  Phagocytosis. — This  theory,  brought 
forward  by  Metchnikoff  to  explain  the  facts  of  natural  and 
acquired  immunity,  has  been  of  enormous  influence  in  stimu- 
lating research  on  the  subject.  Looking  at  the  subject  from  the 
standpoint  of  the  comparative  anatomist,  he  saw  that  it  was  a 
verjf  general  property  possessed  by  certain  cells  throughout 
the  animal  kingdom,  that  they  should  take  up  foreign  bodies 
into  their  interior  and  in  many  cases  digest  and  destroy  them. 
On  extending  his  observations  to  what  occurred  in  disease,  he 
came  to  the  conclusion  that  the  successful  resistance  of  an 
animal  against  bacteria  depended  on  the  activity  of  certain  cells 
called  phagocytes.  In  the  human  subject  he  distinguished  two 
chief  varieties,  namely,  (a)  the  microphages,  which  are  the 
"  polymorpho-nuclear  "  finely  granular  leucocytes  of  the  blood, 
and  (b)  the  macrophages,  which  include  the  larger  hyaline 
leucocytes,  endothelial  cells,  connective  tissue  corpuscles,  and,  in 
short,  any  of  the  larger  cells  which  have  the  power  of  ingesting 
bacteria.  Insusceptibility  to  a  given  disease  is  indicated  by  a 
rapid  activity  on  the  part  of  the  phagocytes,  different  varieties 


496  IMMUNITY 

being  concerned  in  different  cases, — an  activity  which  may 
rapidly  destroy  the  bacteria  and  prevent  even  local  damage.  If 
the  organisms  are  introduced  into  the  tissues  of  a  moderately 
susceptible  animal,  there  occurs  an  inflammatory  reaction  with 
local  leucocytosis,  which  results  in  the  intracellular  destruction 
of  the  invading  organisms.  Phagocytosis  is  regarded  by 
Metchnikoff  as  the  essence  of  inflammation.  He  also  showed 
that  the  bacteria  may  be  in  a  living  and  active  state  when  they 
are  ingested  by  leucocytes.  On  the  other  hand,  he  found  that 
in  a  susceptible  animal  phagocytosis  did  not  occur  or  was  only 
imperfect.  He  also  showed  that  when  a  naturally  susceptible 
animal  was  immunised,  the  process  was  accompanied  by  the 
appearance  of  an  active  phagocytosis.  The  ingestion  of  bacteria 
by  phagocytes  is  undoubtedly  a  phenomenon  of  the  greatest 
importance  in  the  defence  of  the  organism.  It  is  known  that 
amoebae  and  allied  organisms  have  digestive  properties  which 
are  specially  active  towards  bacteria,  and  from  what  can  be 
directly  observed,  as  well  as  indirectly  inferred,  there  can  be  no 
doubt  that  such  a  faculty  is  also  possessed  by  the  phagocytes  of 
the  body.  Thus  bacteria  within  these  cells  are  in  a  position 
favourable  to  their  destruction  and  do  in  many  instances 
become  destroyed.  In  fact,  observations  on  phagocytosis  in 
vitro  show  that  such  destruction  may  in  the  case  of  some 
organisms  occur  so  rapidly  that  the  actual  number  observable  in 
the  leucocytes  is  no  indication  of  the  activity  of  the  process. 
In  other  instances,  e.g.  in  gonorrhoea,  the  ingested  organisms 
would  appear  to  survive  a  considerable  time  without  undergoing 
change.  Undoubtedly  phagocytosis  is  of  the  highest  importance 
in  active  immunity,  as  by  its  means  organisms  which  would  not 
undergo  an  extra-cellular  death  may  be  killed  off.  In  the  process 
of  immunisation  of  a  susceptible  animal  we  see  a  negative  or 
neutral  chemiotaxis  becoming  replaced  by  positive  chemiotaxis. 
This  has  bee  a  explained  by  Metchnikoff  as  due  to  an  education 
or  stimulation  of  the  phagocytes.  The  recent  work  on  opsonins 
shows,  however,  that  this  is  not  the  case,  as  leucocytes  from  an 
immunised  animal  are  not  more  active  in  this  direction  than 
those  of  a  normal  animal,  the  all-important  factor  being  the 
development  of  an  opsonin  in  the  immune  animal.  Thus  this 
phase  of  immunity  comes  to  be  merely  a  part  of  the  subject  of 
anti-substances  in  general. 

The  digestive  ferments  of  phagocytes  or  cytases  are,  according 
to  Metchnikoff,  retained  within  the  cells  under  normal  conditions, 
but  are  set  free  when  these  cells  are  injured,  for  example,  when 
the  blood  is  shed.  They  then  become  free  in  the  serum  by 


THE  THEORY  OF  PHAGOCYTOSIS     497 

the  breaking  up  of  the  cells — the  process  known  as  phagolysis — 
and  they  then  constitute  the  alexines,  or  complements  of  Ehrlich. 
Of  these,  as  has  already  been  said,  Metchnikoff  thinks  there 
are  probably  two  kinds — one  called  macrocytase,  contained  in 
the  macrophages,  which  is  specially  active  toward  the  formed 
elements  of  the  animal  body,  protozoa,  etc. ;  and  the  other, 
microcytase,  contained  within  the  poly morpho-nu clear  leucocytes, 
which  has  a  special  digestive  action  on  bacteria.  It  is  the 
microcytase  which  gives  blood  serum  its  bactericidal  properties. 
It  appears  to  us,  however,  that  Metchnikoff  has  gone  too  far  in 
distinguishing  the  activities  of  the  two  classes  of  cells  so  much 
as  he  has  done. 

When  the  properties  of  antibacterial  sera,  as  above  described, 
are  considered  in  relation  to  phagocytosis,  Metchnikoff  gives  the 
following  explanation.  He  admits  that  the  immune -body  is 
fixed  by  the  bacteria  (or  red  corpuscles,  as  the  case  may  be), 
though  he  does  not  state  that  a  chemical  combination  takes 
place;  hence  he  calls  it  a  fixative  (jfixateur).  The  immune-bodies 
are  to  be  regarded  as  auxiliary  ferments  (ferments  adjuvants) 
which  aid  the  action  of  the  alexine.  Unlike  the  latter,  however, 
they  are  formed  in  excess  during  immunisation  and  set  free  in 
the  serum.  He  compares  their  action  to  that  of  enterokinase,  a 
» ferment  which  is  produced  in  the  intestine  and  which  aids  the 
action  of  trypsin.  Thus,  when  the  bacteria  have  fixed  the  immune- 
body  their  digestion  is  facilitated  either  within  the  phagocytes,  or 
outside  of  them  when  the  alexine  has  been  set  free  by  phagolysis. 
He,  however,  maintains  that  extracellular  digestion  or  lysogenesis 
does  not  take  place  without  the  occurrence  of  phagolysis.  The 
source  of  immune-bodies  is,  in  all  probability,  also  the  leucocytes, 
as  these  substances  are  specially  abundant  in  organs  rich  in 
such  eells — spleen,  lymphatic  glands,  etc. ;  here  again  the  mono- 
nuclear  leucocytes  are  probably  the  source  of  the  immune-bodies 
concerned  in  haemolysis,  the  polymorpho-nuclear  leucocytes  the 
source  of  those  concerned  in  bacteriolysis.  Although  the  im- 
mune-bodies are  usually  set  free  in  the  serum,  this  is  not  always  the 
case  ;  sometimes  they  are  contained  in  the  cells,  and  this  probably 
occurs  when  there  is  a  high  degree  of  active  immunity  against 
bacteria  without  a  serum  having  an  antibacterial  action,  the 
powers  of  intracellular  digestion  being  in  such  cases  increased. 
In  this  way  the  facts  of  immunity  can  be  explained  so  far  as 
these  concern  the  destruction  of  bacteria. 

Metchnikoff's  work  has  less  direct  bearing  on  the  production 
of  antitoxins.  He  admits  the  fixation  of  the  toxin  by  the  anti- 
toxin to  form  a  neutral  compound,  and  he  apparently  considers 
32 


498  IMMUNITY 

that  leucocytes  may  also  be  concerned  in  the  production  of 
antitoxins.  Apart,  however,  from  antitoxin  formation,  he  con- 
siders the  acquired  resistance  of  the  cells  themselves  of  high 
importance  in  toxin  immunity. 

When  we  consider  Metchnikoff's  theory  as  thus  extended  to 
cover  recently  established  facts,  it  must  be  admitted  that  it  affords 
a  rational  explanation  of  a  considerable  part  of  the  subject, 
though  the  elucidation  of  the  chemiotactic  phenomena  during 
immunisation  as  explained  above  detracts  from  the  importance 
which  he  attached  to  the  leucocyte.  It,  however,  does  not  afford 
explanation  of  the  multiplicity  and  specificity  of  antitoxins  as 
Ehrlich's  does  ;  on  the  other  hand,  it  is  more  concerned  with  the 
cells  of  the  body  as  destroyers  or  digesters  of  bacteria.  As 
regards  the  subject  of  antibacterial  sera,  the  results  of  these  two 
workers  may  be  said  to  be  in  harmony  in  some  of  the  funda- 
mental conceptions.  And  it  is  of  interest  to  note  that  Metchni- 
koff,  starting  with  the  phenomena  of  intracellular  digestion,  has 
arrived  at  the  giving  off  of  specific  ferments  by  phagocytes  ;  whilst 
Ehrlich,  from  his  first  investigations  on  the  constitution  of  toxins, 
has  arrived  at  an  explanation  of  antitoxins  and  immune-bodies 
also  with  a  theory  of  cell-nutrition  as  its  basis.  Within  the  last 
few  years  marked  progress  has  thus  been  made  towards  the 
establishment  of  the  fundamental  laws  of  immunity. 

NATURAL  IMMUNITY. 

We  have  placed  the  consideration  of  this  subject  after  that  of 
acquired  immunity,  as  the  latter  supplies  facts  which  indicate  in 
what  direction  an  explanation  of  the  former  may  be  looked  for. 
There  may  be  said  to  be  two  main  facts  with  regard  to  natural 
immunity.  The  first  is,  that  there  is  a  large  number  of  bacteria 
— the  so-called  non-pathogenic  organisms — which  are  practically 
incapable,  unless  perhaps  in  very  large  doses,  of  producing  patho- 
genic effects  in  any  animal ;  when  these  are  introduced  into  the 
body  they  rapidly  die  out.  This  fact,  accordingly,  shows  that 
the  animal  tissues  generally  have  a  remarkable  power  of  destroy- 
ing living  bacteria.  The  second  fact  is,  that  there  are  other 
bacteria  which  are  very  virulent  to  some  species  of  animals, 
whilst  they  are  almost  harmless  to  other  species ;  the  anthrax 
bacillus  may  be  taken  as  an  example.  Now  it  is  manifest  that 
natural  immunity  against  such  an  organism  might  be  due  to  a 
special  power  possessed  by  an  animal  of  destroying  the  organisms 
when  introduced  into  its  tissues.  It  might  also  possibly  be  due 
to  an  insusceptibility  to,  or  power  of  neutralising,  the  toxins  of 


NATURAL   BACTERICIDAL   POWERS  499 

the  organism.  For  the  study  of  the  various  diseases  shows  that 
the  toxins  (in  the  widest  sense)  are  the  weapons  by  which  morbid 
changes  are  produced,  and  that  toxin-formation  is  a  property 
common  to  all  pathogenic  bacteria.  There  is,  moreover,  no 
such  thing  known  as  a  bacterium  multiplying  in  the  living  tissues 
without  producing  local  or  general  changes,  though,  theoretically, 
there  might  be.  As  a  matter  of  fact,  however,  natural  immunity 
is  in  most  cases  one  against  infection,  i.e.  consists  in  a  power 
possessed  by  the  animal  body  of  destroying  the  living  bacteria 
when  introduced  into  its  tissues  :  such  a  power  may  exist  though 
the  animal  is  still  susceptible  to  the  separated  toxins.  We  shall 
now  look  at  these  two  factors  separately. 

1.  Variations  in  Natural  Bactericidal  Powers. — The  funda- 
mental fact  here  is  that  a  given  bacterium  may  be  rapidly 
destroyed  in  one  animal,  whereas  in  another  it  may  rapidly 
multiply  and  produce  morbid  effects.  The  special  powers  of 
destroying  organisms  in  natural  immunity  have  been  ascribed  to 
(a)  phagocytosis,  and  (b)  the  action  of  the  serum. 

(a)  The  chief  factors  with  regard  to  phagocytosis  have  been 
given  above.  The  bacteria  in  a  naturally  immune  animal,  for 
example,  the  anthrax  bacillus  in  the  tissues  of  the  white  rat,  are 
undoubtedly  taken  up  in  large  numbers  and  destroyed  by  the 
phagocytes,  whereas  in  a  susceptible  animal  this  only  occurs  to 
a  small  extent ;  and  Metchnikoff  has  shown  that  they  are  taken 
up  in  a  living  condition,  and  are  still  virulent  when  tested  in  a 
susceptible  animal.  Variations  in  phagocytic  activity  are  found 
to  correspond  more  or  less  closely  with  the  degree  of  immunity 
present,  but  are  probably  in  themselves  capable  of  explanation. 
The  fundamental  observations  of  Wright  and  Douglas  show  that 
in  many  cases  at  least,  leucocytes  do  not  ingest  organisms  in  a 
neutral  saline  solution,  and  that  this  is  not  due  to  the  medium 
in  which  they  are,  is  readily  shown  by  subjecting  the  organisms 
to  the  action  of  fresh  serum  and  then  washing  them ;  thereafter, 
they  are  rapidly  taken  up  by  the  leucocytes  in  salt  solution . 
In  most  cases  this  result  is  due  to  the  labile  opsonin  of  normal 
serum  which  has  combining  affinities  for  a  great  many  organisms 
as  already  stated.  In  other  cases  more  specific  substances  may 
be  concerned.  But  the  all-important  fact  is  that  whether 
phagocytosis  occurs  or  not,  appears  to  depend  upon  certain  bodies 
in  the  serum.  As  yet  we  cannot  say  whether  the  phagocytosis 
in  a  given  serum,  observed  according  to  the  opsonic  technique, 
always  runs  parallel  with  phagocytosis  in  the  tissues  of  the 
animal  from  which  the  serum  has  been  taken.  This  is  a  subject 
on  which  extended  observations  are  necessary.  But  whether  or 


500  IMMUNITY 

not  phagocytosis  in  vivo  corresponds  with  that  in  vitro  it  is 
probably  to  be  explained  in  the  same  way ;  that  is,  it  probably 
depends  upon  the  content  of  the  serum.  The  composition  of  the 
latter,  no  doubt,  is  the  result  of  cellular  activity,  and  in  this 
the  leucocytes  themselves  are  in  all  probability  concerned,  but 
the  movements  and  phagocytic  activity  of  these  cells  seem  to 
be  chiefly  if  not  entirely  controlled  by  their  environments. 
Ingestion  is,  however,  only  the  first  stage  in  the  process ;  intra- 
cellular  destruction  is  the  second,  and  is  of  equal  importance. 
What  may  be  called  intracellular  bactericidal  action  probably 
varies  in  the  case  of  leucocytes  of  different  animals,  but  regarding 
this  our  knowledge  is  deficient,  and,  further,  bacteria  may  some- 
times survive  the  cells  which  have  ingested  them. 

(b)  When  it  had  been  shown  that  normal  serum  possessed 
bactericidal  powers  against  different  organisms,  the  question 
naturally  arose  as  to  whether  this  bactericidal  power  varied  in 
different  animals  in  proportion  to  the  natural  immunity  enjoyed 
by  them.  The  earlier  experiments  of  Behring  appeared  to  give 
grounds  for  the  belief  that  this  was  the  case.  He  found,  for 
example,  that  the  serum  of  the  white  rat,  which  has  a  remark- 
able immunity  to  anthrax,  had  greater  bactericidal  powers  than 
that  of  other  animals  investigated.  Further  investigation,  how- 
ever, has  shown  that  this  is  not  an  example  of  a  general  law, 
and  that  the  bactericidal  action  of  the  serum  does  not  vary  pari 
passu  with  the  degree  of  immunity.  In  many  cases,  however, 
non-pathogenic  and  also  attenuated  pathogenic  bacteria  can  be 
seen  to  undergo  rapid  solution  and  disappear  when  placed  in  a 
drop  of  normal  serum.  The  bactericidal  action  of  the  serum 
was  specially  studied  by  Nuttall,  and  later  by  Buchner  and 
Hankin,  who  believe  that  the  serum  owes  its  power  to  certain 
substances  in  it  derived  from  the  spleen,  lymphatic  glands, 
thymus,  and  other  tissues  rich  in  leucocytes.  To  these 
substances  Buchner  gave  the  name  of  alexines  •  as  already 
explained,  they  correspond  with  MetchnikofPs  cytases  and 
Ehrlich's  complements  described  above.  They  can  be  pre- 
cipitated by  alcohol  and  by  ammonium  sulphate,  and  in  this 
respect  and  in  their  relative  lability  correspond  with  enzymes  or 
unorganised  ferments.  Variations  in  bactericidal  power  of  the 
serum  as  tested  in  vitro,  however,  do  not  explain  the  presence 
or  absence  of  natural  immunity  against  a  living  bacterium.  In 
some  cases,  for  example,  it  has  been  found  to  be  considerable, 
while  the  organisms  flourish  in  the  body,  and  the  animal  has  no 
immunity.  In  such  a  case  Metchnikoff  says  that  there  occurs  in 
the  living  body  no  liberation  of  alexines  by  the  phagocytes,  and 


NATURAL   SUSCEPTIBILITY   TO   TOXINS      501 

hence  no  bactericidal  action  such  as  occurs  when  the  blood  is  shed. 
In  the  case  of  the  haemolytic  action  of  a  normal  serum,  it  has 
been  shown  in  many  instances  that  in  addition  to  complement  a 
natural  immune-body  is  also  concerned  (p.  481),  and  this  would 
appear  to  be  the  rule ;  the  process  being  analogous  to  what  is 
seen  in  the  case  of  an  artificially  developed  hsemolytic  serum. 
In  certain  instances  an  analogous  condition  appears  to  obtain  in 
a  normal  bactericidal  serum.  For  example  the  dog's  serum  heated 
at  58°  C.  contains  a  natural  immune-body  to  anthrax  which  can 
be  activated  by  the  addition  of  normal  guinea-pig's  serum  so  as  to 
produce  a  bactericidal  action,  though  the  latter  is  by  itself  with- 
out any  such  effect.  At  present,  however,  the  possibility  of 
bactericidal  action  by  complement  alone  cannot  be  excluded,  as 
it  appears  to  combine  with  many  bacteria  without  any  inter- 
mediary. Further  work  is  necessary  to  determine  whether  all 
the  facts  regarding  natural  immunity  are  explainable  by  the 
opsonic  and  bactericidal  properties  of  the  serum. 

2.  Variations  in  Natural  /Susceptibility  to  Toxins. — We  must 
here  start  with  the  fundamental  fact,  incapable  of  explanation, 
that  toxicity  is  a  relative  thing,  or  in  other  words,  that  different 
animals  have  different  degrees  of  resistance  or  non-suscepti- 
bility to  toxic  bodies.  In  every  case  a  certain  dose  must  be 
reached  before  effects  can  be  observed,  and  up  to  that  point  the 
animal  has  resistance.  This  natural  resistance  is  found  to 
present  very  remarkable  degrees  of  variation  in  different  animals. 
The  great  resistance  of  the  common  fowl  to  the  toxin  of  the  tetanus 
bacillus  may  be  here  mentioned  (v.  p.  381),  and  large  amounts  of 
this  poison  can  be  injected  into  the  scorpion  without  producing 
any  effects  whatever ;  the  high  resistance  of  the  pigeon  to 
morphia  is,  a  striking  example  in  the  case  of  vegetable  poisons. 
This  variation  in  resistance  to  toxins  applies  also  to  those  which 
produce  local  effects,  as  well  as  to  those  which  cause  symptoms 
of  general  poisoning.  Instances  of  this  are  furnished,  for 
example,  by  the  vegetable  poisons  ricin  and  abrin,  by  the  snake 
poisons,  and  by  bacterial  toxins  such  as  that  of  diphtheria.  We 
must  take  this  natural  resistance  for  granted,  though  it  is 
possible  that  ere  long  it  will  be  explained. 

According  to  Ehrlich's  view  of  the  constitution  of  toxins,  it 
might  be  due  to  the  want  of  combining  affinity  between  the 
tissue  cells  and  the  haptophorous  group  of  the  toxin ;  or,  on  the 
other  hand,  supposing  this  affinity  to  exist,  it  might  be  due  to 
an  innate  non-susceptibility  to  the  action  of  the  toxophorous 
group.  Certain  investigations  have  been  made  in  order  to 
determine  the  combining  affinity  of  the  nervous  system  of  the 


502 


IMMUNITY 


fowl  with  tetanus  toxin,  as  compared  with  that  obtaining  in  a 
susceptible  animal,  but  the  results  have  been  somewhat  contra- 
dictory. Accordingly,  a  general  statement  on  this  point  cannot 
at  present  be  made,  though  in  all  probability  variations  in  the 
susceptibility  to  the  toxophorous  group  will  be  found  to  play  a 
very  important  part.  It  has  been  shown  by  Muir  and  Brown- 
ing by  means  of  hsemolytic  tests  that  the  toxic  activity  of  com- 
plement, after  it  has  been  fixed  to  the  corpuscles,  varies  very 
much ;  in  some  instances  an  amount  of  complement,  which  would 
rapidly  produce  complete  lysis  of  one  kind  of  corpuscle,  may 
have  practically  no  effect  on  another,  even  though  it  enters  into 
combination.  These  results  are  of  importance  in  demonstrating 
how  the  corresponding  molecules  of  different  animals  may  vary 
in  sensitiveness  to  toxic  action. 


APPENDIX  A. 
SMALLPOX  AND  VACCINATION. 

SMALLPOX  is  a  disease  to  which  much  study  has  been  devoted, 
owing,  on  the  one  hand,  to  the  havoc  which  it  formerly  wrought 
among  the  nations  of  Europe — a  havoc  which  at  the  present 
day  it  is  difficult  to  realise, — and  on  the  other  hand,  to  the 
controversies  which  have  arisen  in  connection  with  the  active 
immunisation  against  it  introduced  by  Jenner.  Though  there  is 
little  doubt  that  a  contagium  vivum  is  concerned  in  its  occurrence, 
the  etiological  relationship  of  any  particular  organism  to  smallpox 
has  still  to  be  proved ;  and  with  regard  to  Jennerian  vaccination, 
it  is  only  the  advance  of  bacteriological  knowledge  which  is  now 
enabling  us  to  understand  the  principles  which  underlie  the 
treatment,  and  which  is  furnishing  methods  whereby,  in  the  near 
future,  the  vexed  questions  concerned  will  probably  be  satis- 
factorily settled.  We  cannot  here  do  more  than  touch  on  some 
of  the  results  of  investigation  with  regard  to  the  disease. 

Jennerian  Vaccination. — Up  to  Jenner's  time  the  only  means 
adopted  to  mitigate  the  disease  had  been  by  inoculation  (by 
scarification)  of  virus  taken  from  a  smallpox  pustule,  especially 
from  a  mild  case.  By  this  means  it  was  shown  that  in  the  great 
majority  of  cases  a  mild  form  of  the  disease  was  originated.  It 
had  previously  been  known  that  one  attack  of  the  disease 
protected  against  future  infection,  and  that  the  mild  attack 
produced  by  inoculation  also  had  this  effect.  This  inoculation 
method  had  long  been  practised  in  various  parts  of  the  world, 
and  had  considerable  popularity  all  over  Europe  during  the 
eighteenth  century.  Its  disadvantage  was  that  the  resulting 
disease,  though  mild,  was  still  infectious,  and  thus  might  be  the 
starting-point  of  a  virulent  form  among  unprotected  persons. 
Jenner's  discovery  was  published  wrhen  inoculation  was  still 
considerably  practised.  It  was  founded  on  the  popular  belief 
that  those  who  had  contracted  cowpox  from  an  affected  animal 

503 


504  SMALLPOX   AND   VACCINATION 

were  insusceptible  to  subsequent  infection  from  smallpox.  In  the 
horse  there  occurs  a  disease  known  as  horsepox,  especially  tend- 
ing to  arise  in  wet  cold  springs,  which  consists  in  an  inflammatory 
condition  about  the  hocks,  giving  rise  to  ulceration.  Jenner 
believed  that  the  matter  from  these  ulcers,  when  transferred  by 
the  hands  of  men  who  dressed  the  sores  to  the  teats  of  cows 
subsequently  milked  by  them,  gave  rise  to  cowpox  in  the  latter. 
This  disease  was  thus  identical  with  horsepox  in  epidemics  of 
which  it  had  its  origin.  Jenner  was,  however,  probably  in  error 
in  confounding  horsepox  with  another  disease  of  horses,  namely, 
grease.  Cowpox  manifests  itself  as  a  papular  eruption  on  the 
teats ;  the  papules  become  pustules ;  their  contents  dry  up  to 
form  scabs,  or  more  or  less  deep  ulcers  are  formed  at  their  sites. 
From  such  a  lesion  the  hands  of  the  milkers  may  become  infected 
through  abrasions,  and  a  similar  local  eruption  occurs,  with 
general  symptoms  in  the  form  of  slight  fever,  malaise,  and  loss  of 
appetite.  It  is  this  illness  which,  according  to  Jenner,  gives  rise 
to  immunity  from  smallpox  infection.  He  showed  experimentally 
that  persons  who  had  suffered  from  such  attacks  did  not  react 
to  inoculation  with  smallpox,  and  further,  that  persons  to  whom 
he  communicated  cowpox  artificially,  were  similarly  immune. 
The  results  of  Jenner's  observations  and  experiments  were 
published  in  1798  under  the  title  An  Inquiry  into  the  Causes  and 
Effects  of  the  Variola  Vaccince.  Though  from  the  first  Jennerian 
vaccination  had  many  opponents,  it  gradually  gained  the  con- 
fidence of  the  unprejudiced,  and  became  extensively  practised  all 
over  the  world,  as  it  is  at  the  present  day. 

The  evidence  in  favour  of  vaccination  is  very  strong.  There 
is  no  doubt  that  inoculation  with  lymph  properly  taken  from  a 
case  of  cowpox,  can  be  maintained  with  very  little  variation  in 
strength  for  a  long  time  by  passage  from  calf  to  calf,  and  such 
calves  are  now  the  usual  source  of  the  lymph  used  for  human 
vaccination.  When  lymph  derived  from  them  is  used  for  the 
latter  purpose,  immunity  against  smallpox  is  conferred  on  the 
vaccinated  individual.  It  has  been  objected  that  some  of  the 
lymph  which  has  been  used  has  been  derived  from  calves 
inoculated,  not  with  cowpox,  but  with  human  smallpox.  It  is 
possible  that  this  may  have  occurred  in  some  of  the  strains  of 
lymph  in  use  shortly  after  the  publication  of  Jenner's  discovery, 
but  most  of  the  strains  at  present  in  use  have  probably  been 
derived  originally  from  cowpox.  The  most  striking  evidence  in 
favour  of  vaccination  is  derived  from  its  effects  among  the  staffs 
of  smallpox  hospitals,  for  here,  in  numerous  instances,  it  is  only 
the  unvaccinated  individuals  who  have  contracted  the  disease. 


RELATIONS   OF   SMALLPOX   TO   COWPOX      505 

While  vaccination  is  undoubtedly  efficacious  in  protecting 
against  smallpox,  Jenner  was  wrong  in  supposing  that  a  vaccina- 
tion in  infancy  afforded  protection  for  more  than  a  certain 
number  of  years  thereafter.  It  has  been  noted  in  smallpox 
epidemics  which  have  occurred  since  the  introduction  of  vaccina- 
tion, that  whereas  young  unprotected  subjects  readily  contract 
the  disease,  those  vaccinated  as  infants  escape  more  or  less  till 
after  the  thirteenth  to  the  fifteenth  years.  It  has  become, 
therefore,  more  and  more  evident  that  re  vaccination  is  necessary 
if  immunity  is  to  continue;  and  where  this  is  done  in  any 
population,  smallpox  becomes  a  rare  disease,  as  has  happened  in 
the  German  army,  where  the  mortality  is  practically  nil.  The 
whole  question  of  the  efficacy  of  vaccination  was  investigated  in 
this  country  in  1896  by  a  Royal  Commission,  whose  general 
conclusions  were  as  follows.  Vaccination  diminishes  the  liability 
to  attack  by  smallpox,  and  when  the  latter  does  occur,  the 
disease  is  milder  and  less  fatal.  Protection  against  attack  is 
greatest  during  nine  or  ten  years  after  vaccination.  It  is  still 
efficacious  for  a  further  period  of  five  years,  and  possibly  never 
wholly  ceases.  The  power  of  vaccination  to  modify  an  attack 
outlasts  its  power  wholly  to  ward  it  off.  Revaccination  restores 
protection,  but  this  operation  must  be  from  time  to  time  repeated. 
Vaccination  is  beneficial  according  to  the  thoroughness  with 
which  it  is  performed. 

The  Kelationship  of  Smallpox  (Variola)  to  Cowpox 
(Vaccinia). — This  is  the  question  regarding  which,  since  the 
introduction  of  vaccination,  the  greatest  controversy  has  taken 
place ;  a  subsidiary  point  has  been  the  inter-relationships  within 
the  group  of  animal  diseases  which  includes  cowpox,  horsepox, 
sheep-pox,  and  cattle-plague.  With  reference  to  smallpox  and 
cowpox  the  problem  has  been,  Are  they  identical  or  not  1  There 
is  no  doubt  that  cowpox  can  be  communicated  to  man,  in  whom 
it  produces  the  eruption  limited  to  the  point  of  inoculation,  and 
the  slight  general  symptoms  which  vaccination  with  calf  lymph 
has  made  familiar.  Apparently  against  the  view  that  cowpox  is 
a  modified  smallpox  are  the  facts  that  it  never  reproduces  in 
man  a  general  eruption,  and  that  the  local  eruption  is  only 
infectious  when  matter  from  it  is  introduced  into  an  abrasion. 
The  loss  of  infectiveness  by  transmission  through  the  body  of  a 
relatively  insusceptible  animal  is  a  condition  of  which  we  have 
already  seen  many  instances  in  other  diseases,  and  the  uniformity 
of  the  type  of  the  affection  resulting  from  vaccination  with  calf 
lymph  finds  a  parallel  in  such  a  disease  as  hydrophobia,  where, 
after  passage  through  a  series  of  monkeys,  a  virus  of  attenuated 


506  SMALLPOX   AND   VACCINATION 

but  constant  virulence  can  be  obtained.  We  have  seen  that 
there  are  good  grounds  for  believing  that  the  virus  of  calf  lymph 
confers  immunity  against  human  smallpox.  In  considering  the 
relationships  of  cowpox  and  smallpox,  this  is  an  important 
though  subsidiary  point ;  for  at  present  it  is  questionable 
whether  there  are  any  well -authenticated  instances  of  one 
disease  having  the  capacity  of  conferring  immunity  against 
another.  The  most  difficult  question  in  this  connection  is  what 
happens  when  inoculations  of  smallpox  matter  are  made  on 
cattle.  Chauveau  denies  that  in  such  circumstances  cowpox  is 
obtained.  He,  however,  only  experimented  on  adult  cows.  The 
transformation  has  been  accomplished  by  many  observers, 
including,  in  this  country,  Simpson,  Klein,  Hime,  and  Copeman. 
The  general  result  of  these  experiments  has  been  that  if  a  series 
of  calves  is  inoculated  with  variolous  matter,  in  the  first  there 
may  not  be  much  local  reaction,  though  redness  and  swelling 
appear  at  the  point  of  inoculation,  and  some  general  symptoms 
manifest  themselves.  On  squeezing  some  of  the  lymph  from 
such  reaction  as  occurs,  and  using  it  to  continue  the  passages 
through  other  calves,  after  a  very  few  transfers  a  local  reaction 
indistinguishable  from  that  caused  by  cowpox  lymph  generally 
takes  place,  and  the  animals  are  now  found  to  be  immune 
against  the  latter.  Not  only  so,  but  on  using  for  human 
vaccination  the  lymph  from  such  variolated  calves,  results 
indistinguishable  from  those  produced  by  vaccine  lymph  are 
obtained,  and  the  transitory  illness  which  follows,  unlike  that 
produced  in  man  by  inoculation  with  smallpox  lymph,  is  no 
longer  infectious.  In  fact,  many  of  the  strains  of  lymph  in  use 
in  Germany  at  present  have  been  derived  thus  from  the  variola- 
tion  of  calves.  ,  The  criticism  of  these  experiments  which  has 
been  offered,  namely,  that  since  many  of  them  were  performed 
in  vaccine  establishments,  the  calves  were  probably  at  the  same 
time  infected  with  vaccinia,  is  not  of  great  weight,  as  in  all  the 
recent  cases  at  least,  very  elaborate  precautions  have  been 
adopted  against  such  a  contingency.  And  at  any  rate  it  would 
be  rather  extraordinary  that  this  accident  should  happen  to 
occur  in  every  case.  We  can,  therefore,  say  that  at  present 
there  is  the  very  strongest  ground  for  holding  not  only  that 
vaccinia  confers  immunity  against  variola,  but  that  variola 
confers  immunity  against  vaccinia.  The  experimentum  crucis 
for  establishing  the  identity  of  the  two  diseases  would  of  course 
be  the  isolation  of  the  same  micro-organism  from  both,  and  the 
obtaining  of  all  the  results  just  detailed  by  means  of  pure 
cultures  or  the  products  of  such.  In  the  absence  of  this 


BACTERIA   IN   SMALLPOX  507 

evidence  we  are  at  present  justified  in  considering  that  there  is 
strong  reason  for  believing  that  vaccinia  and  variola  are  the  same 
disease,  and  that  the  differences  between  them  result  from  the 
relative  susceptibilities  of  the  two  species  of  animals  in  which 
they  naturally  occur. 

With  regard  to  the  relation  of  cowpox  to  horsepox,  it  is 
extremely  probable  that  they  are  the  same  disease.  Some 
epidemics  of  the  former  have  originated  from  the  horse,  but  in 
other  cases  such  a  source  has  not  been  traced.  Cattle-plague 
from  the  clinical  standpoint,  and  also  from  that  of  pathological 
anatomy,  resembles  very  closely  human  smallpox.  Though 
each  of  the  two  diseases  is  extremely  infectious  to  its  appropriate 
animal,  there  is  no  record  of  cattle-plague  giving  rise  to  small- 
pox in  man  or  vice  versd.  When  matter  from  a  cattle-plague 
pustule  tis  inoculated  in  man,  a  pustule  resembling  a  vaccine 
pustule  occurs,  and  further,  the  individual  is  asserted  to  be  now 
immune  to  vaccination ;  but  vaccination  of  cattle  with  cowpox 
lymph  offers  no  protection  against  cattle-plague,  though  some 
have  looked  on  the  latter  as  merely  a  malignant  cowpox.  Sheep- 
pox  also  has  many  clinical  and  pathological  analogies  with 
human  smallpox,  and  facts  as  to  its  relation  to  cowpox  vaccina- 
tion similar  to  those  observed  in  cattle -plague  have  been 
reported.  Smallpox,  cowpox,  cattle-plague,  horsepox,  and  sheep- 
pox,  in  short,  constitute  an  interesting  group  of  analogous 
diseases,  of  the  true  relationships  of  which  to  one  another  we 
are,  however,  still  ignorant. 

Micro-organisms  associated  with  Smallpox. — Burdon  Sander- 
son was  among  the  first  to  show  that  in  vaccine  lymph  there 
were  certain  bodies  which  he  recognised  as  bacteria.  Since 
then  numerous  observations  have  been  made  as  to  the  occurrence 
of  such  in  matter  derived  from  variolous  and  vaccine  pustules. 
In  especially  the  later  stages  of  the  latter,  many  of  the  pyogenic 
organisms  are  always  present,  e.g.  staphylococcus  aureus  and 
staphylococcus  cereus  flavus,  and  many  of  the  ordinary  skin 
saprophytes  also  are  often  present,  but  no  organism  has  ever 
been  isolated  which  on  transference  to  animals  has  been  shown 
to  have  any  specific  relationship  to  the  disease.  Streptococci 
have  also  been  described  as  agglutinable  by  the  sera  of  smallpox 
patients  and  of  vaccinated  persons ;  such  sera  it  may  be  said 
had  no  effect  on  other  strains  of  streptococci.  Calmette  and  Guerin 
have  described  very  minute  granules  in  the  lymph  which  could 
not  be  cultivated  but  which  persisted  after  all  the  bacteria  had 
been  removed.  (The  method  by  which  the  latter  was  accom- 
plished was  by  exciting  a  leucocytosis  in  a  rabbit's  peritoneum  and 


508  SMALLPOX  AND   VACCINATION 

then  introducing  the  vaccinal  lymph  ;  the  leucocytes  phagocyted 
the  bacteria  so  that  the  lymph  no  longer  gave  cultures  on  ordinary 
media.  It  was,  however,  still  potent  to  produce  vaccinia.) 

Klein  and  also,  independently,  Copeman,  have  observed  an  organism 
in  lymph  taken  from  a  vaccine  pustule  in  a  calf  on  the  fifth  and  sixth 
days,  in  human  vaccine  lymph  on  the  eighth  day,  and  in  lymph  from 
a  smallpox  pustule  on  the  fourth  day.  To  demonstrate  the  bacilli, 
cover-glass  films  are  dried  and  placed  for  five  minutes  in  acetic  acid  (1 
in  2),  washed  in  distilled  water,  dried,  and  placed  in  alcoholic  gentian- 
violet  for  from  twenty-four  to  forty-eight  hours,  after  which  they  are 
washed  in  water  and  mounted.  Copeman  and  Kent  also  found  the 
bacilli  in  sections  of  vaccine  pustules  stained  by  Loffler's  methylene-blue, 
or  by  Gram's  method.  The  organisms  are  '4  to  '8  /u.  in  length,  and 
one-third  to  a  half  of  this  in  thickness.  They  are  generally  thinner  and 
stain  better  at  the  ends  than  at  the  middle.  They  occur  in  groups  of 
from  three  to  ten  in  both  the  lymph  and  the  tissues.  In  the  centre  of 
their  protoplasm  there  is  often  a  clear  globule,  which  is  looked  on  as  a 
spore.  They  have  hitherto  resisted  the  ordinary  isolation  methods,  a 
fact  which  is  rather  in  favour  of  their  non-saprophytic  nature.  By 
inoculating  fresh  eggs  with  the  crusts  of  smallpox  pustules  Copeman  has, 
however,  obtained  a  growth  of  a  bacillus  resembling  that  found  by  him 
in  the  tissues.  Though  subcultures  on  ordinary  media  have  been 
obtained,  the  pathogenic  effects  of  these  have  not  been  fully  investigated, 
and  thus  the  identity  of  this  bacillus  with  that  seen  in  the  tissues  is 
not  proved. 

Various  observers  have  described  appearances  in  the  epithelial 
cells  in  the  neighbourhood  of  the  smallpox  or  vaccine  pustules, 
which  they  have  interpreted  as  being  protozoa.  Thus  Ruffer 
and  Plimmer  describe  as  occurring  in  clear  vacuoles  in  the  cells 
of  the  rete  Malpighii  at  the  edge  of  the  pustule,  in  paraffin 
sections  of  vaccine  and  smallpox  pustules  carefully  hardened  in 
alcohol,  and  stained  by  the  Ehrlich-Biondi  mixture,  small  round 
bodies  of  about  four  times  the  size  of  a  staphylococcus  pyogenes, 
coloured  red  by  the  acid  fuchsin,  sometimes  with  a  central  part 
stained  by  the  methyl-green.  These  are  described  as  multiplying 
by  simple  division,  and  in  the  living  condition  exhibiting 
amoeboid  movement.  Similar  bodies  have  been  described  by 
Reed  in  the  blood  of  smallpox  patients  and  of  vaccinated 
children  and  calves. 

These  are  probably  the  bodies  described  by  Guarnieri  and  to 
which  considerable  attention  has  been  paid.  They  are  from 
1-8  ft  in  diameter,  are  round,  oval,  or  sickle -shaped,  and  stain 
by  ordinary  nuclear  dyes.  They  lie  in  the  cells  in  spaces 
often  near  the  nucleus,  and  are  readily  demonstrable  in  vaccine 
pustules  and  also  in  the  experimental  lesions  which  can  be 
produced  in  the  rabbit's  cornea,  the  larger  bodies  being  denned 
in  the  cells  towards  the  centre  of  the  lesion.  These  bodies 


NATURE   OF   VACCINATION  509 

have  been  looked  on  by  many  as  protozoa,  and  Guarnieri  himself 
stated  that  multiplication  could  be  seen  occurring  in  them  in 
fresh  lymph,  but  Ewing  and  also  Prowazek  have  brought  forward 
strong  evidence  for  the  appearances  being  due  to  nuclear  changes, 
Still  the  question  of  the  specificity  of  these  changes  to  variolous 
lesions  remains,  and  here  it  may  be  said  Wasielewski  has  shown 
that  they  persist  through  46  transfers  on  the  cornea  of  the 
rabbit  and  further  no  similar  appearances  have  been  found  in 
other  skin  lesions.  Prowazek  examined  material  fixed  in  a  hot 
mixture  of  two- thirds  saturated  perchloride  of  mercury  and 
one -third  90  per  cent,  alcohol,  washed  in  40  per  cent,  iodine 
alcohol  and  stained  in  Grenadier's  haematoxylin,  and  found 
bodies  in  the  epithelial  cells  1-4  /*,  in  size,  sharply  contoured 
and  having  ragged  edges  as  if  made  up  of  massed  chromosomes. 
These  were  often  broader  at  one  end  than  at  the  other,  and 
appearances  have  been  seen  which  suggest  longitudinal  division. 
Prowazek  has  also  seen  these  "  lymph-bodies  "  as  he  has  called 
them  in  the  lymph,  and  he  inclines  to  the  idea  that  they  may  be 
protozoa.  Bonhoff  and  also  Carini  have  described  spirochsetes 
as  occurring  in  variolous  lesions,  but  this  has  not  been  confirmed. 
Future  investigations  must  show  what  significance  is  to  be 
attached  to  these  various  observations. 

The  causal  organism  of  smallpox  is  probably  very  small  as, 
though  there  has  been  some  difference  in  opinion  on  this  point, 
there  is  little  doubt  that  it  will  pass  through  the  coarser  porcelain 
filters. 

The  Nature  of  Vaccination. — As  we  are  ignorant  of  the  cause 
of  smallpox,  we  can  only  conjecture  what  the  nature  of  vaccina- 
tion is.  From  what  we  know  of  other  like  processes,  however, 
we  have  some  ground  for  believing  that  it  consists  in  an  active 
immunisation  by  means  of  an  attenuated  form  of  the  causal 
organism.  As  to  how  immunity  is  maintained  after  vaccination, 
we  do  not  know  much.  Some,  including  Beclere,  Chambon, 
and  Menard  (who  jointly  investigated  the  subject),  maintain  that 
in  the  blood  of  vaccinated  animals  substances  exist  which,  when 
transferred  to  other  animals,  can  confer  a  certain  degree  of 
passive  immunity  against  vaccination,  and  which  have  also  a 
degree  of  curative  action  in  animals  already  vaccinated.  Beumer 
and  Peiper,  on  the  other  hand,  could  not  find  evidence  of  the 
existence  of  such  bodies. 


APPENDIX   B. 
HYDROPHOBIA, 

SYNONYMS. RABIES  :    FRENCH,  LA  RAGE  :    GERMAN,  LYSSA, 

DIE  HUNDSWUTH,  DIE  TOLLWUTH. 

Introductory. — Hydrophobia  is  an  infectious  disease  which  in 
nature  occurs  epidemically  chiefly  among  the  carnivora,  especi- 
ally in  the  dog  and  the  wolf.  Infection  is  carried  by  the  bite 
of  a  rabid  animal  or  by  a  wound  or  abrasion  being  licked  by 
such.  The  disease  can  be  transferred  to  other  species,  and 
when  once  started  can  be  spread  from  individual  to  individual 
by  the  same  paths  of  infection.  Thus  it  occurs  epidemically 
from  time  to  time  in  cattle,  sheep,  horses,  and  deer,  and  can  be 
communicated  to  man.  It  is  questionable  whether  infection 
can  take  place  from  man  to  man  as  the  saliva  of  a  person 
suffering  from  hydrophobia  appears  not  to  contain  the  virus. 
It  is  to  be  noted  that  the  virus  is  apparently  extremely  potent, 
as  cases  of  infection  taking  place  through  an  unabraded  mucous 
membrane  by  the  licking  of  a  rabic  animal  are  on  record  and  the 
experimental  applications  of  the  virus  to  such  surfaces  as  the 
mucous  membrane  of  the  nose  or  the  conjunctiva  is  often  followed 
by  infection. 

In  Western  Europe  the  disease  is  most  frequently  observed 
in  the  dog ;  but  in  Eastern  Europe,  especially  in  Russia, 
epidemics  among  wolves  constitute  a  serious  danger  both  to 
other  animals  and  to  man.  All  the  manifestations  of  the  disease 
point  to  a  serious  affection  of  the  nervous  system  ;  but  inasmuch 
as  symptoms  of  excitement  or  of  depression  may  predominate, 
it  is  customary  to  describe  clinically  two  varieties  of  rabies,  (1) 
rabies  proper,  or  furious  rabies  (la  rage  vraie,  la  rage  furieuse : 
die  rasende  Wuth)  ;  and  (2)  dumb  madness  or  paralytic  rabies  (la 
rage  mue  :  die  stille  Wuth).  The  disease,  however,  is  essentially 
the  same  in  both  cases.  In  the  dog  the  furious  form  is  the 

510 


PATHOLOGY   OF   HYDROPHOBIA  511 

more  common.  After  a  period  of  incubation  of  from  three  to 
six  weeks,  the  first  symptom  noticed  is  a  change  in  the  animal's 
aspect ;  it  becomes  restless,  it  snaps  at  anything  which  it  touches, 
and  tears  up  and  swallows  unwonted  objects ;  it  has  a  peculiar 
high-toned  bark.  Spasms  of  the  throat  muscles  come  on, 
especially  in  swallowing,  and  there  is  abundant  secretion  of 
saliva ;  its  supposed  special  fear  of  water  is,  however,  a  myth, 
— it  fears  to  swallow  at  all.  Gradually  convulsions,  paralysis, 
and  coma  come  on ;  and  death  supervenes.  In  the  paralytic 
form,  the  early  symptoms  are  the  same,  but  paralysis  appears 
sooner.  The  lower  jaw  of  the  animal  drops,  from  implication 
of  the  elevator  muscles,  all  the  muscles  of  the  body  become 
more  or  less  weakened,  and  death  ensues  without  any  very 
marked  irritative  symptoms. 

In  man  the  incubation  period  after  infection  varies  from 
fifteen  days  to  seven  or  eight  months,  or  even  longer,  but  is 
usually  about  forty  days.  When  symptoms  of  rabies  are  about 
to  appear,  certain  prodromata,  such  as  pains  in  the  wound  and 
along  the  nerves  of  the  limb  in  which  the  wound  has  been 
received,  may  be  observed.  To  this  succeeds  a  stage  of  nervous 
irritability,  during  which  all  the  reflexes  are  augmented — the 
victim  starting  at  the  slightest  sound,  for  example.  There  are 
spasms,  especially  of  the  muscles  of  deglutition  and  respiration, 
and  cortical  excitement  evidenced  by  delirium  may  occur.  On 
this  follows  a  period  in  which  all  the  reflexes  are  diminished, 
weakness  and  paralysis  are  observed,  convulsions  occur,  and 
finally  coma  and  death  supervene.  The  duration  of  the  acute 
illness  is  usually  from  four  to  eight  days,  and  death  invariably 
results.  The  existence  of  paralytic  rabies  in  man  has  been 
denied  by  some,  but  it  undoubtedly  occurs.  This  is  usually 
manifested  by  paralysis  of  the  limb  in  which  the  infection  has 
been  received,  and  of  the  neighbouring  parts  ;  but  while  in  such 
cases  this  is  often  the  first  symptom  observed,  during  the  whole 
of  the  illness  the  occurrence  of  widespread  and  progressive 
paralysis  is  the  outstanding  feature.  In  man  then  also  occur 
cases  where  the  cerebellum  and  also  the  sympathetic  system  seem 
to  be  specially  affected. 

The  Pathology  of  Hydrophobia.— In  hydrophobia  as  in 
tetanus,  to  which  it  bears  more  than  a  superficial  resemblance, 
the  appearances  presented  in  the  nervous  system,  to  which  all 
symptoms  are  naturally  referred,  are  comparatively  unimportant. 
On  naked-eye  examination,  congestions,  and,  it  may  be,  minute 
haemorrhages  in  the  central  nervous  system,  are  the  only  features 
noticeable.  Microscopically,  leucocytic  exudation  into  the  peri- 


512  HYDROPHOBIA 

vascular  lymphatic  spaces  in  the  nerve  centres  has  been  observed, 
and  in  the  cells  of  the  anterior  cornua  of  the  grey  matter  in  the 
spinal  cord,  and  also  in  the  nuclei  of  the  cranial  nerves,  various 
degenerations  have  been  described.  Round  the  nerve  cells  in 
the  grey  matter  of  the  cord  and  medulla  Babes  described 
accumulations  of  newly-formed  cells,  and  Van  Gehuchten  observed 
a  phagocytosis  of  the  cells  in  the  posterior  root  ganglia  and  also 
in  the  sympathetic  ganglia.  Both  of  these  conditions  were  at  one 
time  thought  to  be  specific  of  rabies,  but  this  has  been  found  not 
to  be  the  case.  In  the  white  matter,  especially  in  the  posterior 
columns,  swelling  of  the  axis  cylinders  and  breaking  up  of  the 
myeline  sheaths  have  been  noted,  and  similar  changes  occur  also 
in  the  spinal  nerves,  especially  of  the  part  of  the  body  through 
which  infection  has  come.  In  the  nervous  system  also  some 
have  seen  minute  bodies  which  they  have  considered  to  be  cocci, 
but  there  is  no  evidence  that  they  are  really  of  this  nature.  The 
changes  in  the  other  parts  of  the  body  are  unimportant. 

Experimental  pathology  confirms  the  view  that  the  nervous 
system  is  the  centre  of  the  disease  by  finding  in  it  a  special 
concentration  of  what,  from  want  of  a  more  exact  term,  we  must 
call  the  hydrophobic  virus.  Earlier  inoculation  experiments 
made  by  subcutaneous  injection  of  material  from  various  parts 
of  animals  dead  of  rabies  had  not  given  uniform  results,  as, 
whatever  was  the  source  of  the  material,  the  disease  was  not 
invariably  produced.  Pasteur's  first  contribution  to  the  subject 
was  to  show  that  the  most  certain  method  of  infection  was  by 
inserting  the  infective  matter  beneath  the  dura  mater.  He 
found  that  in  the  case  of  any  animal  or  man  dead  of  the  disease, 
injection,  by  this  method,  of  emulsions  of  any  part  of  the  central 
nervous  system,  of  the  cerebro-spinal  fluid,  or  of  the  saliva, 
invariably  gave  rise  to  rabies,  and  also  that  the  natural  period  of 
incubation  was  shortened.  Further,  the  identity  of  the  furious 
and  paralytic  forms  was  proved,  as  sometimes  the  one,  sometimes 
the  other,  was  produced,  whatever  form  had  been  present  in  the 
original  case.  Inoculation  into  the  anterior  chamber  of  the  eye 
is  nearly  as  efficacious  as  subdural  infection.  Infection  with  the 
blood  of  rabic  animals  does  not  reproduce  the  disease.  There  is 
evidence,  however,  that  the  poison  also  exists  in  such  glands  as 
the  pancreas  and  mamma.  Subcutaneous  infection  with  part  of 
the  nervous  system  of  an  animal  dead  of  rabies  usually  gives 
rise  to  the  disease. 

In  consequence  of  the  introduction  of  such  reliable  inoculation 
methods,  further  information  has  been  acquired  regarding  the 
spread  and  distribution  of  the  virus  in  the  body.  Gaining 


THE  VIRUS   OF   HYDROPHOBIA  513 

entrance  by  the  infected  wound,  it  early  manifests  its  affinity  for 
the  nervous  tissues.  It  reaches  the  central  nervous  system 
chiefly  by  spreading  up  the  peripheral  nerves.  This  can  be 
shown  by  inoculating  an  animal  subcutaneously  in  one  of  its 
limbs,  with  virulent  material.  If  now  the  animal  be  killed 
before  symptoms  have'  manifested  themselves,  rabies  can  be 
produced  by  subdural  inoculation  from  the  nerves  of  the  limb 
which  was  infected.  Further,  rabies  can  often  be  produced  from 
such  a  case  by  subdural  infection  with  the  part  of  the  spinal 
cord  into  which  these  nerves  pass,  while  the  other  parts  of  the 
animal's  nervous  system  do  not  give  rise  to  the  disease.  This 
explains  how  the  initial  symptoms  of  the  disease  (pains  along 
nerves,  paralysis,  etc.)  so  often  appear  in  the  infected  part  of  the 
body,  and  it  probably  also  explains  the  fact  that  bites  in  such 
richly  nervous  parts  as  the  face  and  head  are  much  more  likely 
to  be  followed  by  hydrophobia  than  bites  in  other  parts  of  the 
body.  Again,  injection  into  a  peripheral  nerve,  such  as  the 
sciatic,  is  almost  as  certain  a  method  of  infection  as  injection 
into  the  subdural  space,  and  gives  rise  to  the  same  type  of 
symptoms  as  injection  into  the  corresponding  limb.  Intravenous 
injection  of  the  virus,  on  the  other  hand,  differs  from  the  other 
modes  of  infection  in  that  it  more  frequently  gives  rise  to 
paralytic  rabies.  This  fact  Pasteur  explained  by  supposing 
that  the  whole  of  the  nervous  system  in  such  a  case  becomes 
simultaneously  affected.  In  certain  animals  the  virus  seems  to 
have  an  elective  affinity  for  the  salivary  glands,  as  well  as  for 
the  nervous  system.  Roux  and  Nocard  found  that  the  saliva  of 
the  dog  became  virulent  three  days  before  the  first  appearance 
of  symptoms  of  the  disease. 

The  Virus  of  Hydrophobia. — While  a  source  of  infection 
undoubtedly  occurs  in  all  cases  of  hydrophobia,  and  can  usually 
be  traced,  all  attempts  to  determine  the  actual  morbific  cause 
have  been  unsatisfactory.  In  this  connection  various  organisms 
have  been  described  as  being  associated  with  the  disease. 

Thus  Memmo  has  isolated  an  organism  which  resembles  a  yeast,  but 
which  he  places  amongst  the  blastomycetes,  and  with  which  he  states 
he  has  produced  both  types  of  rabies  in  rabbits  and  dogs.  Bruschettini 
also,  by  using  media  containing  brain  substance,  has  grown  a  bacillus 
having  some  resemblances  to  the  members  of  the  diphtheria  group,  and 
with  which  he  claims  to  have  produced  paralytic  rabies  in  rabbits.  In 
the  case  of  the  work  of  neither  of  these  observers  has  there  been  con- 
firmation from  independent  sources,  and  in  neither  case  is  there  evidence 
of  the  crucial  test  having  been  applied,  namely,  that  of  immunising 
animals  against  the  ordinary  hydrophobic  virus  by  means  of  pure 
cultures  of  the  alleged  causal  organism.  With  regard  to  other  possible 

33 


514  HYDROPHOBIA 

causal  agents,  Grigorjew  thinks  such  may  be  found  in  a  protozoon  which 
he  has  constantly  observed  after  inoculation  in  the  cornea. 

In  1903  Negri  described  certain  bodies  as  occurring  in  the 
nervous  system  in  animals  dying  of  rabies  to  which  considerable 
attention  has  since  been  devoted,  and  regarding  the  significance 
of  which  opinion  is  still  divided.  It  may  be  said  that  Negri's 
observations  have  been  generally  confirmed,  and  as  it  is  probable, 
whatever  the  final  opinion  as  to  the  nature  of  the  bodies 
may  be,  that  their  occurrence  is  specific  to  the  disease  and  hence 
may  be  used  for  diagnosis,  we  shall  describe  the  methods  for 
their  demonstration.  In  doing  so  we  shall  chiefly  follow  the 
work  of  the  American  observers,  Williams  and  Lowden,  who, 
more  than  any  others  who  have  confirmed  Negri,  have  used 
methods  widely  employed  in  the  investigation  of  similar 
appearances. 

Their  chief  method  is  to  take  a  piece  of  the  brain  tissue,  to  squeeze  it 
between  a  slide  and  cover-glass,  and,  sliding  off  the  latter,  to  make  a 
smear  which  is  then  fixed  in  methyl  alcohol  for  five  minutes  and  stained 
by  Giemsa's  stain  (p.  107)  for  half  an  hour  to  three  hours  ;  the  prepara- 
tion is  then  washed  in  tap  water  for  2-3  min.  and  dried.  For  rapid  work, 
after  fixation,  equal  parts  of  distilled  water  and  stain  are  used  instead  of 
the  more  dilute  mixture. 

For  sections  the  tissues  are  left  in  Zenker's  fluid  x  for  3-4  hours,  then 
placed  in  tap  water  for  five  minutes,  80  per  cent  alcohol  with  enough 
iodine  added  to  give  it  a  port  wine  colour  for  24  hours  ;  95  per  cent 
alcohol  and  iodine,  24  hours  ;  absolute  alcohol,  4-6  hours  ;  cleared  with 
cedar  oil  and  embedded  in  paraffin  of  melting  point  52°  C.  ;  sections  should 
b3  3-6  yu,  thick.  For  staining,  Mallory's  methylene  -  blue  eosin  is 
recommended  ;  the  steps  are  as  follows  :  xylol ;  absolute  alcohol ;  95 
per  cent  alcohol  and  iodine,  |  hour  ;  95  per  cent  alcohol,  |  hour ; 
absolute  alcohol,  ^'hour  ;  eosin  solution  (5-10  per  cent  aqueous  solution), 
2)  minutes  ;  rinse  in  tap  water  ;  Unua's  polychrome  methylene -blue 
solution  diluted  1-4  with  distilled  water,  15  minutes  ;  differentiation  in 
95  per  cent  alcohol  for  1-5  minutes  (the  preparation  being  kept  in 
motion  and  its  progress  watched  with  a  low  power)  ;  rapid  and  careful 
dehydration  and  clearing. 

The  bodies  vary  much  in  size,  measuring  from  '5  ft  to  25  /z. 
They  are  round,  oval,  or  angular  in  outline.  They  are  found  in 
the  protoplasm  of  the  nerve  cells  and  of  their  processes.  They 
have  a  hyaline  appearance  with  a  sharply-defined  outline,  and  in 
their  substance  they  contain  granular  material.  Taking  for  granted 
their  cellular  structure  we  may  say  that  with  the  Giemsa  mixture 

1  Zenker's  fluid  is  of  the  following  composition  :  potassium  bichromate 
2 '5  gr.,  sodium  sulphate  1  gr.,  perchloride  of  mercury  5  gr.,  glacial  acetic 
acid  5  c.c.,  water  to  100  c.c.  Dissolve  the  perchloride  of  mercury  and  the 
bichromate  of  potassium  in  the  water  with  the  aid  of  heat  and  add  the 
acetic  acid. 


THE   VIRUS   OF   HYDROPHOBIA  515 

their  cytoplasm  stains  blue  and  the  granules  a  blue-red, — by 
Mallory's  stain  the  cytoplasm  is  magenta  and  the  granules  a 
deep  blue.  The  cytoplasm  is  homogeneous,  and  in  it  is  a 
nucleus -like  body  whose  chromatin  particles  in  the  larger 
individuals  are  arranged  round  the  periphery,  there  being  a 
clear  centre  containing  a  nucleolus  ;  in  the  smaller  forms  the 
nucleus  is  a  mere  chromatin  spot.  Round  the  central  definite 
nuclear  body  are  some  chromatoid  particles  which  are  irregular 
in  outline  and  size,  are  sometimes  elongated,  and  do  not  take 
on  such  a  pure  chromatin  stain  as  the  nucleus.  There  is 
evidence  of  division  of  the  nucleus,  and  sometimes  there  may 
apparently  be  three  or  four  nuclei  in  one  body  without  division 
of  the  protoplasm  having  occurred.  Sometimes  the  chromatin 
appears  to  fragment  and  break  up  into  a  large  number  of  small 
particles,  and  in  such  bodies  active  budding  of  the  protoplasm 
may  be  seen.  Sometimes  the  bodies  seem  to  go  on  dividing 
again  and  again,  with  the  result  that  some  very  small  forms  may 
be  produced,  these  sometimes  appearing  in  mulberry  masses. 

The  Negri  bodies  have  been  found  in  nearly  all  cases  of 
street-rabies  examined  by  many  observers,  and  have  never  been 
found  in  other  conditions  of  brain  disease.  They  occur  in  all 
parts  of  the  central  nervous  system,  but  are  said  to  be  most 
abundant  in  the  cells  of  the  cornu  Ammonis.  They  are 
apparently  not  so  readily  found,  at  least  in  their  larger  forms, 
in  animals  dying  from  the  inoculation  of  virus  fixe.  What  the 
significance  of  these  bodies  is,  it  is  at  present,  impossible  to  say ; 
but  whatever  may  be  their  nature,  there  is  now  considerable 
evidence  that  their  presence  is  specific  of  rabies,  and  that  thus 
in  their  recognition  a  much  quicker  means  of  diagnosis  is  possible 
than  by  the  longer  method  of  awaiting  symptoms  in  an  in- 
oculated rabbit.  Many  have  looked  on  these  bodies  as  protozoa, 
and  their  appearance  is  not  inconsistent  with  such  a  view.  The 
objection  which  has  been  raised,  that  if  they  were  protozoa 
they  could  not  pass  through  a  porcelain  filter  (vide  infra}  as 
the  virus  does,  is  met  by  the  fact  of  the  occurrence  of  minute 
forms,  and  by  the  fact  that  similar  small  forms  probably  exist 
in  certain  trypanosomes  (see  Appendix  E).  The  occurrence  of 
minute  forms  would  also  account  for  the  non-recognition  of  the 
parasite  in  the  more  acute  forms  of  the  disease  where  there  had 
been  an  active  vegetative  condition,  and  thus  no  time  for  the 
larger  forms  to  develop. 

There  is  no  doubt  that  between  rabies  and  the  bacterial 
diseases  we  have  studied  there  are  at  every  point  analogies,  the 
most  striking  being  the  protective  inoculation  methods  which 


516  HYDKOPHOBIA 

constitute  the  great  work  of  Pasteur ;  and  everything  points  to 
a  micro-organism  being  the  cause.  The  organism,  whatever  it  is, 
is,  in  its  infective  form,  probably  very  small,  as  it  can  pass 
through  the  coarser  Berkefeld  filters,  and  also  occasionally 
through  the  coarser  Chamberland  candles.  Evidence  that  it  is 
the  organism  itself  which  passes  through,  is  found  in  the  fact 
that  when  an  animal  dies  from  infection  with  the  filtrate,  a 
small  portion  of  its  central  nervous  system  will  originate  the 
disease  in  a  fresh  animal.  Judging  from  our  knowledge  of 
similar  diseases  we  would  strongly  suspect  that  it  is  actually 
present  in  a  living  condition  in  the  central  nervous  system,  the 
saliva,  etc.,  which  yield  what  we  have  called  the  hydrophobic 
virus,  for  by  no  mere  toxin  could  the  disease  be  transmitted 
through  a  series  of  animals,  as  we  shall  presently  see  can  be 
done.  A  toxin  may,  however,  be  concerned  in  the  production 
of  the  pathogenic  effects.  Remlinger  found  that  death  with 
paralytic  symptoms  sometimes  followed  the  injection  of  filtered 
virus,  but  that  the  nervous  system  of  the  dead  animals  did  not 
reproduce  rabies.  He  explains  this  occurrence  by  supposing  that 
the  filtrate  contained  a  toxin  but  not  the  actual  infective  agent. 
The  resistance  of  the  virus  to  external  agents  varies.  Thus  a 
nervous  system  containing  it  is  virulent  till  destroyed  by  putre- 
faction ;  it  can  resist  the  prolonged  application  of  a  temperature 
of  from  -  10°  to  -  20°  C.,  but,  on  the  o£her  hand,  it  is  rendered 
non-virulent  by  one  hour's  exposure  at  50°  C.  Again,  its 
potency  probably  varies  in  nature  according  to  the  source. 
Thus,  while  the  death-rate  among  persons  bitten  by  mad  dogs  is 
about  16  per  cent,  the  corresponding  death-rate  after  the  bites 
of  wolves  is  80  per  cent.  Here,  however,  it  must  be  kept  in 
view  that,  as  the  wolf  is  naturally  the  more  savage  animal,  the 
number  and  extent  of  the  bites,  i.e.  the  number  of  channels  of 
entrance  of  the  virus  into  the  body,  and  the  total  dose,  are 
greater  than  in  the  case  of  persons  bitten  by  dogs.  As  we  shall 
see,  alterations  in  the  potency  of  the  virus  can  certainly  be 
effected  by  artificial  means. 

The  Prophylactic  Treatment  of  Hydrophobia. — Until  the 
publication  of  Pasteur's  researches  in  1885,  the  only  means 
adopted  to  prevent  the  development  of  hydrophobia  in  a  person 
bitten  by  a  rabid  animal  had  consisted  in  the  cauterisation  of 
the  wound.  Such  a  procedure  was  undoubtedly  not  without 
effect.  It  has  been  shown  that  cauterisation  within  five  minutes 
of  the  infliction  of  a  rabic  wound  prevents  the  disease  from 
developing,  and  that  if  done  within  half  an  hour  it  saves  a 
proportion  of  the  cases.  After  this  time,  cauterisation  only 


PROPHYLACTIC  TREATMENT  OF  HYDROPHOBIA  517 

lengthens   the   period   of    incubation ;    but,    as   we    shall    see 
presently,  this  is  an  extremely  important  effect. 

The  work  of  Pasteur  has,  however,  revolutionised  the  whole 
treatment  of  wounds  inflicted  by  hydrophobic  animals.  Pasteur 
started  with  the  idea  that,  since  the  period  of  incubation  in  the 
case  of  animals  infected  subdurally  from  the  nervous  systems  of 
mad  dogs  is  constant  in  the  dog,  the  virus  has  been  from  time 
immemorial  of  constant  strength.  Such  a  virus,  of  what  might 
be  called  natural  strength,  is  usually  referred  to  in  his  works  as 
the  virus  of  la  rage  des  rues,  in  the  writings  of  German  authors 
as  the  virus  of  die  Strasswuth.  Pasteur  found  on  inoculating  a 
monkey  subdurally  with  such  a  virus,  and  then  inoculating 
a  second  monkey  from  the  first,  and  so  on  with  a  series  of 
monkeys,  that  it  gradually  lost  its  virulence,  as  evidenced  by 
lengthened  periods  of  incubation  on  subdural  inoculation  of 
dogs,  until  it  wholly  lost  the  power  of  producing  rabies  in  dogs, 
when  introduced  subcutaneously.  When  this  point  had  been 
attained,  its  virulence  was  not  diminished  by  further  passage 
through  the  monkey.  On  the  other  hand,  if  the  virus  of  la 
rage  des  rues  were  similarly  passed  through  a  series  of  rabbits 
or  guinea-pigs,  its  virulence  wTas  increased  till  a  constant  strength 
(the  virus  fixe)  was  attained.  Pasteur  had  thus  at  command 
three  varieties  of  virus — that  of  natural  strength,  that  which  had 
been  attenuated,  and  that  which  had  been  exalted.  He  further 
found  that,  commencing  with  the  subcutaneous  injection  of  a 
weak  virus  and  following  this  up  with  the  injection  of  the 
stronger  varieties,  he  could  ultimately,  in  a  very  short  time, 
immunise  dogs  against  subdural  infection  with  a  virus  which, 
under  ordinary  conditions,  would  certainly  have  caused  a  fatal 
result.  He  also  elucidated  the  fact  that  the  exalted  virus  con- 
tained in  the  spinal  cords  of  rabbits  such  as  those  referred  to, 
could  be  attenuated  so  as  no  longer  to  produce  rabies  in  dogs  by 
subcutaneous  injection.  This  was  done  by  drying  the  cords  in 
air  over  caustic  potash  (to  absorb  the  moisture),  the  diminution 
of  virulence  being  proportional  to  the  length  of  time  during 
which  the  cords  were  kept.  Accordingly,  by  taking  a  series  of 
such  spinal  cords  kept  for  various  periods  of  time,  he  was 
supplied  with  a  series  of  vaccines  of  different  strengths.  Pasteur 
at  once  applied  himself  to  find  whether  the  comparatively  long 
period  of  incubation  in  man  could  not  be  taken  advantage  of  to 
"  vaccinate  "  him  against  the  disease  before  its  gravest  manifesta- 
tion took  place.  The  following  is  the  record  of  the  first  case 
thus  treated.  The  technique  was  to  rub  up  in  a  little  sterile 
bouillon  a  small  piece  of  the  cord  used,  and  inject  it  under  the 


518 


HYDROPHOBIA 


skin  by  means  of  a  hypodermic  syringe.  The  first  injection  was 
made  with  a  very  attenuated  virus,  i.e.  a  cord  fourteen  days  old. 
In  subsequent  injections  the  strength  of  the  virus  was  gradually 
increased,  as  shown  in  the  table  :— 


uly  7,  1885,  9  A.M.,  cord  of  June  23,  i 

e.  14  days  old. 

7 

6  P.M.      „     25 

12 

8 

9  A.M.     „     27 

11 

8 

6  P.M.     ,,     29 

9 

9 

11  A.M.,  cord  of  July  1 

8 

10 

3 

7 

11 

5 

6 

12 

7 

5 

13 

9 

4 

14 

11 

3 

15 

13 

2 

16 

15 

1  day  old. 

The  patient  never  manifested  the  slightest  symptom  of  hydro- 
phobia. Other  similarly  favourable  results  followed ;  and  this 
prophylactic  treatment  of  the  disease  quickly  gained  the  con- 
fidence of  the  scientific  world,  which  it  still  maintains.  (The 
principal  is,  of  course,  the  same  as  in  artificially  developing  a 
high  degree  of  active  immunity  against  a  bacterial  infection.) 

The  only  modification  which  the  method  has  undergone  has  been  in 
the  treatment  of  serious  cases,  such  as  multiple  bites  from  wolves, 
extensive  bites  about  the  head,  especially  in  children,  cases  which  come 
under  treatment  at  a  late  period  of  the  incubation  stage,  and  cases  where 
the  wounds  have  not  cicatrised.  In  such  cases  the  stages  of  the  treat- 
ment are  condensed.  Thus  on  the  first  day,  say  at  11  A.M.  and  4  P.M. 
and  9  P.M.,  cords  of  12,  10,  and  8  days  respectively  are  used  ;  on  the 
second  day,  cords  of  6,  4,  and  2  days  ;  on  the  third  day,  cords  of  1  day  ; 
on  the  fourth  day,  cords  of  8,  6,  and  4  days  ;  on  the  fifth,  cords  of  3  and 
2  days  ;  on  the  sixth,  cords  of  1  day  ;  and  so  on  for  ten  days.  In  each 
case  the  average  dose  is  about  2  c.c.  of  the  emulsion. 

The  success  of  the  treatment  has  been  very  marked.  The  statistics 
of  the  cases  treated  in  Paris  are  published  quarterly  in  the  Annalcs  de 
rinstitut  Pasteur,  and  general  summaries  of  the  results  of  each  year 
are  also  prepared.  As  we  have  said,  the  ordinary  mortality  formerly 
was  16  per  cent  of  all  persons  bitten.  During  the  ten  years  1886-95, 
17,337  cases  were  treated,  with' a  mortality  of  '48  per  cent.  It  has  been 
alleged  that  many  people  are  treated  who  have  been  bitten  by  dogs  that 
were  not  mad.  This,  however,  is  not  more  true  of  the  cases  treated  by 
Pasteur's  method  than  it  was  of  those  on  which  the  ordinary  mortality 
of  16  per  cent  Avas  based,  and  care  is  taken  in  making  up  the  statistics 
to  distinguish  the  cases  into  three  classes.  Class  A  includes  only  persons 
bitten  by  dogs  proved  to  have  had  rabies,  by  inoculation  in  healthy 
animals  of  parts  of  the  central  nervous  system  of  the  diseased  animal. 
Class  B  includes  those  bitten  by  dogs  that  a  competent  veterinary  surgeon 
has  pronounced  to  be  mad.  Class  C  includes  all  other  cases.  During 
1895,  122  cases  belonging  to  Class  A  were  treated,  with  no  deaths  ;  940 
belonging  to  class  B,  with  two  deaths  ;  and  449  belonging  to  Class  C, 


METHODS  519 

with  no  deaths.  Besides  the  Institute  in  Paris,  similar  institutions  exist 
in  other  parts  of  France,  in  Italy,  and  especially  in  Russia,  as  well  as 
in  other  parts  of  the  world  ;  and  in  these  similar  success  has  been 
experienced.  It  may  be  now  taken  as  established,  that  a  very  grave 
responsibility  rests  on  those  concerned,  if  a  person  bitten  by  a  mad 
animal  is  not  subjected  to  the  Pasteur  treatment.  Sometimes  during  or 
after  treatment  there  appear  slight  paralytic  symptoms  with  obstinate 
constipation  and  it  may  he  retention  of  urine,  but  these  pass  off  within 
a  few  weeks  and  leave  behind  no  ill  effects. 

Antirabic  Serum, — In  the  early  part  of  the  nineteenth  century 
an  Italian  physician,  Valli,  showed  that  immunity  against  rabies 
could  be  conferred  by  administering  through  the  stomach  pro- 
gressively increasing  doses  of  hydrophobic  virus.  Following  up 
this  observation,  Tizzoni  and  Centanni  have  attenuated  rabic 
virus  by  submitting  it  to  peptic  digestion,  and  have  immunised 
animals  by  injecting  gradually  increasing  strengths  of  such  virus. 
This  method  is  usually  referred  to  as  the  Italian  method  of 
immunisation.  The  latter  workers  showed  from  this  that  the 
serum  of  animals  thus  immunised  could  give  rise  to  passive 
immunity  in  other  animals ;  and  further,  that  if  injected  into 
animals  from  seven  to  fourteen  days  after  infection  with  the 
virus,  it  prevented  the  latter  from  producing  its  fatal  effects, 
even  when  symptoms  had  begun  to  manifest  themselves.  They 
further  succeeded  in  producing  in  the  sheep  and  the  dog  an 
immunity  equal  to  from  1-25,000  to  1-50,000  (vide  p.  454),  and 
they  recommended  the  use,  in  severe  cases,  of  the  serum  of  such 
animals  in  addition  to  the  treatment  of  the  patient  by  the 
Pasteur  method.  A  like  serum  has  been  obtained  from  animals 
treated  by  the  ordinary  Pasteur  method. 

Methods,  (a)  Diagnosis. — When  a  person  is  bitten  by  an 
animal  suspected  to  be  rabid,  the  latter  must  under  no  circum- 
stances be  killed.  Much  more  can  be  learned  by  watching  it 
while  alive  than  by  post-mortem  examination.  In  the  latter  case 
only  such  things  as  the  occurrence  of  broken  teeth,  marked 
congestion  of  the  fauces,  or  the  presence  of  unwonted  material 
in  the  stomach  throw  any  light  on  the  condition ;  nothing  of  a 
positive  nature  can  be  learned  from  examining  the  nervous 
system.  On  the  other  hand,  in  the  living  animal  the  develop- 
ment of  the  characteristic  symptoms  can  be  watched,  and  death 
will  occur  in  not  more  than  five  days.  If  the  suspected  animal 
has  been  killed,  then  a  small  piece  of  its  medulla  or  cord  must 
be  taken,  with  all  aseptic  precautions,  rubbed  up  in  a  little 
sterile  '75  per  cent  sodium  chloride  solution,  and  injected  by 
means  of  a  syringe  beneath  the  dura  mater  of  a  rabbit,  the  latter 
having  been  trephined  over  the  cerebrum  by  means  of  the  small 


520  HYDROPHOBIA 

trephine  which  is  made  for  the  purpose.  Symptoms  usually 
occur  in  from  ten  to  twenty-three  days  and  death  in  fifteen  to 
twenty-five  days.  When  such  inoculation  has  to  be  practised  it 
is  evident  that  the  diagnosis  is  delayed.  When  the  material  for 
inoculation  has  to  be  sent  any  distance  this  is  best  effected  by 
packing  the  head  of  the  animal  in  ice.  The  virulence  of  organs 
is  not  lost,  however,  if  they  are  simply  placed  in  sterile  water  or 
glycerin  in  well-stoppered  bottles.  When  the  brain  of  the 
suspected  dog  is  available  either  through  its  death  or  its  being 
killed,  the  Negri  bodies  should  be  sought  for  especially  in  the 
cornu  Ammonis  by  the  methods  described  above. 

(b)  Treatment. — Every  wound  inflicted  by  a  rabid  animal 
ought  to  be  cauterised  with  the  actual  cautery  as  soon  as  possible. 
By  such  treatment  the  incubation  period  will  at  any  rate  be 
lengthened,  and  therefore  there  will  be  better  opportunity  for 
the  Pasteur  inoculation  method  being  efficacious.  The  person 
ought  then  to  be  sent  to  the  nearest  Pasteur  Institute  for  treat- 
ment. It  is  of  great  importance  that  in  such  a  case  the  nervous 
system  of  the  animal  should  also  be  sent,  in  order  that  the 
diagnosis  may  be  certainly  verified. 


APPENDIX  C. 

MALARIAL  FEVER. 

IT  has  now  been  conclusively  proved  that  the  cause  of  malarial 
fever  is  a  protozoon  of  which  there  are  several  species.  They 
belong  to  the  hsemosporidia  (a  sub-class  of  the  sporozoa)  which 
are  blood  parasites,  infecting  the  red  corpuscles  of  mammals, 
reptiles,  and  birds.  The  parasite  was  formerly  known  as  the 
hcematozoon  or  plasmodium  malarice,  although  the  use  of  the 
latter  term  is  incorrect ;  the  term  hcemamoeba  is,  however,  now 
generally  employed.  The  parasite  was  first  observed  by  Laveran 
in  1880,  and  his  discovery  received  confirmation  from  the  in- 
dependent researches  of  Marchiafava  and  Celli,  and  later  from 
the  researches  of  many  others  in  various  parts  of  the  world. 
Golgi  supplied  valuable  additional  information,  especially  in 
relation  to  the  sporulation  of  the  organism  and  the  varieties  in 
different  types  of  malarial  fever.  In  this  country  valuable  work 
on  the  subject  was  done  by  Manson,  and  to  him  specially  belongs 
the  credit  of  regarding  the  exflagellation  of  the  organism  as 
a  preparation  for  an  extra-corporeal  phase  of  existence.  By  in- 
duction he  arrived  at  the  belief  that  the  cycle  of  existence  outside 
the  human  body  probably  took  place  in  the  mosquito.  It  was 
specially  in  order  to  discover,  if  possible,  the  parasite  in  the 
mosquito,  that  Ross  commenced  his  long  series  of  observations, 
which  were  ultimately  crowned  with  success.  After  patient  and 
persistent  search,  he  found  rounded  pigmented  bodies  in  the 
wall  of  the  stomach  of  a  dapple-winged  mosquito  (a  species  of 
Anopheles)  which  had  been  fed  on  the  blood  of  a  malarial 
patient.  The  pigment  in  .these  bodies  was  exactly  similar  to 
that  in  the  malarial  parasite,  and  he  excluded  the  possibility  of 
their  representing  anything  else  than  a  stage  in  the  life  cycle  of 
the  organism.  He  confirmed  this  discovery  and  obtained  cor- 
responding results  in  the  case  of  the  proteosoma  infection  of 
birds,  where  the  parasite  is  closely  related  to  that  of  malaria. 

521 


522  MALARIAL   FEVER 

In  birds  affected  with  this  organism,  he  was  able  to  trace  all 
stages  of  its  development,  from  the  time  it  entered  the  stomach 
along  with  the  blood,  till  the  time  when  it  settled  in  a  special 
form  in  the  salivary  glands  of  the  insect.  Ross's  results  were 
published  in  1898.  Exactly  corresponding  stages  were  after- 
wards found  in  the  case  of  the  different  species  of  the  human 
parasite,  by  Grassi,  Bignami,  and  Bastianelli ;  and  these  with 
other  Italian  observers  also  supplied  important  information 
regarding  the  transmission  of  the  disease  by  infected  mosquitoes. 
Abundant  additional  observations,  with  confirmatory  results, 
were  supplied  by  Koch,  Daniels,  Christophers,  Stephens,  and 
others.  Wherever  malaria  has  been  studied  the  result  has  been 
the  same.  Lastly,  we  may  mention  the  striking  experiment 
carried  out  by  Manson  by  means  of  mosquitoes  fed  on  the  blood 
of  patients  in  Italy  suffering  from  mild  tertian  fever.  The 
insects,  after  being  thus  fed,  were  taken  to  London  and  allowed 
to  bite  the  human  subject,  Manson's  son,  Dr.  P.  Thurburn 
Manson,  offering  himself  for  the  purpose.  The  result  was  that 
infection  occurred ;  the  parasites  appeared  in  the  blood,  and 
were  associated  with  an  attack  of  tertian  fever.  Ross's  discovery 
has  not  only  been  a  means  of  elucidating  the  mode  of  infection, 
but,  as  will  be  shown  below,  has  also  supplied  the  means  of 
successfully  combating  the  disease. 

From  the  zoological  point  of  view  the  mosquito  is  regarded 
as  the  definitive  host  of  the  parasite,  the  human  subject  as  the 
intermediate  host.  But  in  describing  the  life  history,  it  will  be 
convenient  to  consider,  first,  the  cycle  in  the  human  body,  and, 
secondly,  that  in  the  mosquito.  Various  terms  have  been 
applied  to  the  various  stages,  but  we  shall  give  those  now 
generally  used. 

The  Cycle  in  the  Human  Subject. — With  regard  to  this 
cycle,  it  may  be  stated  that  the  parasite  is  conveyed  by  the  bite 
of  the  mosquito  in  the  form  of  a  small  filamentous  cell — 
sporozoite  or  exotospore,  which  penetrates  a  red  corpuscle  and 
becomes  a  small  amoeboid  organism  or  amoebula.  There  is  then 
a  regularly  repeated  asexual  cycle  of  the  parasite  in  the  blood, 
the  length  of  which  cycle  determines  the  type  of  the  fever. 
During  this  cycle  there  is  a  growth  of  the  amcebulae  or 
trophozoites  within  the  red  corpuscles  up  to  their  complete 
development ;  sporulation  or  schizogony  then  occurs.  The  onset 
of  the  febrile  attack  corresponds  with  the  stage  of  sporulation 
and  the.  setting  free  of  the  spores  (enhsemospores  or  merozoites), 
i.e.  with  the  production  of  a  fresh  brood  of  parasites.  These 
spores  soon  become  attached  to,  and  penetrate  into  the  interior 


FORMS   OF   THE   MALARIAL   PARASITE       523 

of  the  red  corpuscles,  becoming  intra-corpuscular  amoebuke  ;  the 
cycle  is  thus  completed.  The  parasites  are  most  numerous  in 
the  blood  during  the  development  of  the  pyrexia,  and,  further, 
they  are  also  much  more  abundant  in  the  internal  organs  than  in 
the  peripheral  blood ;  in  the  malignant  type,  for  example,  the 
process  of  sporulation  is  practically  confined  to  the  former. 

In  addition  to  these  forms  which  are  part  of  the  ordinary 
asexual  cycle,  there  are  derived  from  the  amoebulse  other  forms, 
which  are  called  gametocytes,  or  sexual  cells.  These  remain 
unaltered  during  successive  attacks  of  pyrexia,  and  undergo  no 
further  change  until  the  blood  is  removed  from  the  human  body. 
In  the  simple  tertian  and  quartan  fevers  (vide  infra)  the  gameto- 
cytes  resemble  somewhat  in  appearance  the  fully  developed 
amcebulse  before  sporulation,  whereas  in  the  malignant  type  they 
have  a  characteristic  crescent-like  or  sausage-shaped  form  ;  hence 
they  are  often  spoken  of  as  "crescentic  bodies." 

The  various  forms  of  the  parasite  seen  in  the  human  blood 
may  now  be  described  more  in  detail. 

1.  The    Enhcemospores    (Lankester)    or   Merozoites   are   the 
youngest  and  smallest  forms  resulting  from  the  segmentation  of 
the  adult  amoebula — sporocyte  or  schizont.     They  are  of  round 
or  oval  shape  and  of  small  size,  usually  not  exceeding  2  fj.  in 
diameter ;  the  size,  however,  varies  somewhat  in  the  different 
types  of  fever.      A  nucleus  and  peripheral  protoplasm  can  be 
distinguished    (Fig.    159).       The    former    appears   as   a    small 
rounded  body  which  usually  remains  unstained,  but  contains  a 
minute   mass   of  chromatin  which  stains  a  deep  red  with  the 
Romano wsky  method ;    the  peripheral  protoplasm  is  coloured 
fairly  deeply  with  methylene-blue.     The  spores  show  little  or 
no  amoeboid  movement ;  at  first  free  on  the  plasma,  they  soon 
attack  the  red  corpuscles,  where  they  become  the  intra-corpuscular 
amcebulte.     If  the  blood,  say  in  a  mild  tertian  case,  be  examined 
in  the  early  stages  of  pyrexia,  one  often  finds  at  the  same  time 
sporulating  forms,  free  spores,  and  the  young  amoebulse  within 
the  red  corpuscles. 

2.  Intra-corpuscular  Amoebulce  or  Trophozoites. — These  include 
the  parasites  which  have  attacked  the  red  corpuscles  ;  they  are  at 
first  situated  on  the  surface  of  the  latter  but  afterwards  penetrate 
their  substance.     They  usually  occur  singly  in  the  red  corpuscles, 
but  sometimes  two  or  more  may  be  present  together.       The 
youngest  or  smallest  forms  appear  as  minute  colourless  specks, 
of  about  the  same  size  as  the  spores.     As  seen  in  fresh  blood, 
they  exhibit  more  or  less  active  amoeboid  movement,  showing 
marked  variations  in  shape.     The  amount  and  character  of  the 


524  MALARIAL   FEVER 

amoeboid  movement  varies  somewhat  in  different  types  of  fever. 
As  they  increase  in  size,  pigment  appears  in  their  interior  as 
minute  dark  brown  or  black  specks,  and  gradually  becomes 
more  abundant  (Figs.  155,  156).  The  pigment  may  be 
scattered  through  their  substance,  or  concentrated  at  one  or 
more  points,  and  often  shows  vibratory  or  oscillating  movements. 
This  pigment  is  elaborated  from  the  haemoglobin  of  the 
red  corpuscles,  the  parasite  growing  at  the  expense  of  the  latter. 
The  red  corpuscles  thus  invaded  may  remain  unaltered  in 
appearance  (quartan  fever),  may  become  swollen  and  pale  (tertian 
fever),  or  somewhat  shrivelled  and  of  darker  tint  (malignant 
fever).  In  stained  specimens  a  nucleus  may  be  seen  in  the 
parasite  as  a  pale  spot  containing  chromatin  which  may  be 
arranged  as  a  single  concentrated  mass  or  as  several  separated 
granules,  the  chromatin  being  coloured  a  deep  red  by  the 
Romanowsky  method.  The  protoplasm  of  the  parasite,  which 
is  coloured  of  varying  depth  of  tint  with  methylene-blue,  shows 
great  variation  in  configuration  (Fig.  156).  The  young  parasites 
not  unfrequently  present  a  "ring-form,"  a  portion  of  the  red 
corpuscle  being  thus  enclosed  by  the  parasite.  These  ring-forms 
are  met  with  in  all  the  varieties  of  the  parasite,  but  they  are 
especially  common  in  the  case  of  the  malignant  parasite,  where 
they  are  of  smaller  size  and  of  more  symmetrical  form  than  in 
the  others  (Fig.  160);  the  pigment  is  usually  collected  in  a 
small  clump  at  one  side. 

Within  the  red  corpuscles  the  parasites  gradually  increase 
in  size  till  the  full  adult  form  is  reached  (Fig.  157).  In  this 
stage  the  parasite  loses  its  amoeboid  movement  more  or  less 
completely,  has  a  somewhat  rounded  form,  and  contains  a 
considerable  amount  of  pigment.  In  the  malignant  form  it  only 
occupies  a  fraction  of  the  red  corpuscle.  The  adult  parasites 
may  then  undergo  sporulation,  but  not  all  of  thejn  do  so ;  some 
become  degenerated  and  ultimately  break  down. 

3.  Sporocytes  or  Schizonts. — In  the  process  of  schizogony 
the  chromatin  becomes  divided  into  a  number  of  daughter 
nuclei  which  are  scattered  through  the  protoplasm  ;  the  latter 
then  undergoes  corresponding  segmentation  and  the  small 
merozoites  or  enhsemospores  result.  The  pigment  during  the 
process  becomes  aggregated  in  the  centre  and  is  surrounded  by 
a  small  quantity  of  residuary  protoplasm.  Schaudinn  has  found 
in  the  case  of  the  tertian  parasite  that  schizogony  begins  by  a  sort 
of  primitive  mitosis,  which  is  then  followed  by  simple  multiple 
fission.  The  spores  or  merozoites  are  of  rounded  or  oval  shape, 
as  above  described,  and  are  set  free  by  the  rupture  of  the 


FIG.  154. 


FIG.  155. 


FIG.  156. 


FIG.  157. 


f. 


FIG.  158.  FIG.  159. 

FIGS.  154-159. — Various  phases  of  the  benign  tertian  parasite. 

Fig.  154.  Several  young  ring-shaped  amoebulse  within  the  red  corpuscles,  one  of  the 
latter  enlarged  and  showing  a  dotted  appearance.  Fig.  155.  A  larger  amcebula  con- 
taining pigment  granules.  Fig.  156.  Two  large  amcebulfe,  exemplifying  the  great 
variation  in  form.  Fig.  157.  Large  amoebula  assuming  the  spherical  form  and  showing 
isolated  fragments  of  chromatin — preparatory  to  sporulation.  Fig.  153.  Sporocyte 
or  schizont,  which  has  produced  eighteen  spores,  each  of  which  contains  a  small 
collection  of  chromatin.  Fig.  159.  A  number  of  spores  which  have  just  been  set 
free  in  the  plasma,  x  1000. 


FIG.  160. 


FIG.  161, 


FIG.  162. 


FIG.  163. 


: 


FIG.  164.  FIG.  165. 

FIGS.  160-165.— Exemplifying  phases  of  the  malignant  parasite. 

Fig.  160.  Two  small  ring-shaped  amoebulte  within  the  red  corpuscles.  Fig.  161.  A 
"crescent"  or  gamete  showing  the  envelope  of  the  red  corpuscles;  also  an  amoebula. 
Figs.  162-165  illustrate  the  changes  in  form  undergone  by  the  crescents  outside  the 
body.  In  the  interior  of  the  spherical  form  in  Fig.  164  evidence  of  the  flagella  can  be 
seen.  Fig.  165.  A  male  gametocyte  which  has  undergone  exflagellation,  showing  the 
thread-like  microgametes  or  spermatozoa  attached  at  the  periphery.  x.1000.  (The 
figures  in  this  plate  are  from  preparations  kindly  lent  by  Sir  Patrick  Manson.) 


FORMS   OF   THE   MALARIAL   PARASITE       527 

envelope  of  the  red  corpuscle.  The  pigment  also  becomes  free 
and  may  be  taken  up  by  leucocytes.  The  number  and  arrange- 
ment of  the  spores  within  the  sporocyte  vary  in  the  different 
types.  In  the  quartan  there  are  6-12,  and  the  segmentation  is 
in  a  radiate  manner,  giving  rise  to  the  characteristic  daisy-head 
appearance ;  in  the  tertian  they  number  1 5-20  or  more,  and 
have  a  somewhat  rosette-like  arrangement  (Fig.  158);  in  the 
malignant  there  are  usually  6-12  spores  of  small  size  and 
somewhat  irregularly  arranged. 

Gametocytes. — As  stated  above,  these  are  sexual  cells  which 
are  formed  from  certain  of  the  amcebulce,  and  which  undergo 
no  further  development  in  the  human  subject.  In  the  mild 
tertian  and  quartan  fevers  they  are  rounded  and  resemble  some- 
what the  largest  amoebulse.  The  female  cells,  macrogametocytes, 
are  of  large  size,  measuring  up  to  1 6  ^  in  diameter ;  they  con- 
tain coarse  grains  of  pigment,  and  the  protoplasm  stains  somewhat 
deeply  with  methylene-blue.  The  male  cells,  microgametocytes, 
are  smaller,  and  the  protoplasm  stains  faintly ;  the  nucleus, 
generally  in  the  centre,  is  rich  in  chromatin.  In  the  malignant 
fevers  the  gametocytes  have  the  special  crescentic  form  mentioned 
above.  They  measure  8-9  p  in  length,  and  occasionally  a  fine 
curved  line  is  seen  joining  the  extremities  on  the  concave  aspect, 
which  represents  the  envelope  of  the  red  corpuscle  (Fig.  161). 
They  are  colourless  and  transparent,  and  are  enclosed  by  a 
distinct  membrane ;  in  the  central  part  there  is  a  collection  of 
pigment,  and  granules  of  chromatin.  It  is  stated  that  the  male 
crescents  can  be  distinguished  from  the  female  by  their  appear- 
ance. In  the  former  the  pigment  is  less  dark  and  more  scattered 
through  the  cell,  and  there  are  several  granules  of  chromatin ; 
in  the  latter  the  pigment  is  dark  and  concentrated,  often  in  a 
small  ring,  and  there  are  one  or  two  masses  of  chromatin  in  the 
centre  of  the  crescent.  According  to  the  Italian  observers  the 
early  forms  of  the  crescents  are  somewhat  fusiform  in  shape  and 
are  produced  in  the  bone-marrow.  The  fully  developed  crescents 
do  not  appear  in  the  blood  till  several  days  after  the  onset  of 
the  fever,  and  they  may  be  found  a  considerable  time  after  the 
disappearance  of  the  pyrexial  attacks.  They  are  also  little,  if  at 
all,  influenced  by  the  administration  of  quinine. 

It  is  well  known  that  after  a  patient  has  apparently  recovered 
from  malarial  fever  a  relapse  may  take  place  without  fresh 
infection  occurring,  and  Schaudinn  has  published  interesting 
observations  bearing  on  this  point.  He  has  found  that  the 
inacrogametocyte  of  tertian  fever  may  by  a  process  of  partheno- 
genesis give  rise  to  merozoites,  which  in  their  turn  infect  the  red 


528  MALARIAL   FEVER 

corpuscles  and  start  the  cycle  again.  As  described  and  figured 
by  him,  the  chromatin  of  the  macrogametocyte  divides  first  into 
two  portions,  one  of  which  is  smaller  and  stains  more  deeply 
than  the  other.  This  more  deeply-staining  portion  then  divides, 
and  the  protoplasm  becomes  segmented  as  in  ordinary  schizogony, 
and  a  young  brood  of  parasites  results.  The  more  faintly-staining 
chromatin  along  with  part  of  the  protoplasm  breaks  up  and 
disappears. 

The  Cycle  in  the  Mosquito. — As  already  explained,  this 
starts  from  the  gametocytes.  After  the  blood  is  shed,  or  after 
it  is  swallowed  by  the  mosquito,  two  important  phenomena 
occur,  viz.  (a)  the  full  development  of  the  sexual  cells  or 
gametocytes,  and  (6)  the  impregnation  of  the  female.  If  the 
blood  from  a  case  of  malignant  infection  be  examined  in  a  moist 
chamber,  preferably  on  a  warm  stage,  under  the  microscope, 
both  male  and  female  gametocytes  may  be  seen  to  become  oval 
and  afterwards  rounded  in  shape  (Figs.  162-164).  Thereafter, 
in  the  case  of  the  male  cell,  a  vibratile  or  dancing  movement  of 
the  pigment  granules  can  be  seen  in  the  interior,  and  soon 
several  flagella-like  structures  shoot  out  from  the  periphery 
(Fig.  165).  They  are  of  considerable  length  but  of  great  fineness, 
and  often  show  a  somewhat  bulbous  extremity.  By  the 
Romanowsky  method  they  have  been  found  to  contain  a  delicate 
core  of  chromatin,  which  is  covered  by  protoplasm.  They 
represent  the  male  cells  proper,  that  is,  they  are  sperm -cells  or 
spermatozoa ;  they  are  also  known  as  microgametes.  They 
become  detached  from  the  sphere  and  move  away  in  the 
surrounding  fluid.  The  female  cell  also  assumes  the  rounded 
form,  and  maturation  takes  place  by  the  giving  off  of  part  of 
the  nuclear  chromatin.  Impregnation  occurs  by  the  entrance  of 
a  microgamete,  the  chromatin  of  the  two  cells  afterwards 
becoming  fused.  Impregnation  was  first  observed  by  MacCallum 
in  the  case  of  halteridium,  and  he  found  that  the  female  cell 
afterwards  acquired  the  power  of  independent  movement  or 
became  a  "travelling  vermicule."  He  also  observed  the 
impregnation  of  the  malignant  parasite.  The  fertilised  female 
cell  is  now  generally  spoken  of  as  a  zygote  or  ookinete. 

It  has  been  established  that  the  phenomena  just  described 
occur  within  the  stomach  of  the  mosquito,  and  that  the  fertilised 
cell  or  zygote  penetrates  the  stomach  wall  and  settles  between 
the  muscle  fibres ;  on  the  second  day  after  the  mosquito  has 
ingested  the  infected  blood  small  rounded  cells  about  6-8  //,  in 
diameter  and  containing  clumps  of  pigment  may  be  found  in 
this  position.  (It  was  in  fact  the  character  of  the  pigment 


VARIETIES   OF   THE   MALARIAL   PARASITE     529 

which  led  Ross  to  believe  that  he  had  before  him  a  stage  in  the 
development  of  the  malarial  parasite.)  A  distinct  membrane 
called  a  sporocyst  forms  around  the  zygote,  and  on  subsequent 
days  a  great  increase  in  size  takes  place,  and  the  cysts  come  to 
project  from  the  surface  of  the  stomach  into  the  body  cavity. 
The  zygote  divides  into  a  number  of  cells  called  blastopkores  or 
sporoblasts,  and  these  again  divide  and  form  a  large  number  of 
filiform  cells  which  have  a  radiate  arrangement;  these  were 
called  by  Ross  "  germinal  rods,"  but  are  now  usually  known  as 
sporozoites  or  exotospores  (in  contradistinction  to  the  enhaemospores 
of  the  human  cycle).  The  full  development  within  the  sporocyst 
occupies,  in  the  case  of  proteosoma,  about  seven  days,  in  the 
case  of  the  malarial  parasites  a  little  longer.  When  fully 
developed  the  cyst  measures  about  60  /x  in  diameter,  and  appears 
packed  with  sporozoites.  It  then  bursts,  and  the  latter  are  set 
free  in  the  body  cavity.  A  large  number  settle  within  the 
large  veneno-salivary  gland  of  the  insect,  and  are  thus  in  a 
position  to  be  injected  along  with  its  secretion  into  the  human 
subject.  The  sporozoites  enter  red  corpuscles  and  become 
amcebulse  as  above  described.  Daniels  found  that  in  the  case 
of  the  malignant  parasite  an  interval  of  twelve  days  at  least 
intervened  between  the  time  of  feeding  the  mosquito  and  the 
appearance  of  the  sporozoites  in  the  gland. 

It  will  thus  be  seen  that  in  the  human  subject  the  parasite 
passes  through  an  indefinite  number  of  regularly  recurring  asexual 
cycles,  with  the  giving  off  of  collateral  sexual  cells,  and  that  in 
the  mosquito  there  is  one  cycle  which  may  be  said  to  start  with 
the  impregnation  of  the  female  gamete. 

Varieties  of  the  Malarial  Parasite. — The  view  propounded 
by  Laveran  was  that  there  is  only  one  species  of  malarial  parasite, 
which  is  polymorphous,  and  presents  slight  differences  in 
structural  character  in  the  different  types  of  fever.  It  may, 
however,  now  be  accepted  as  proved  that  there  are  at  least  three 
distinct  species  which  infect  the  human  subject.  Practically  all 
are  agreed  as  to  a  division  into  two  groups,  one  of  which 
embraces  the  parasites  of  the  milder  fevers — "  winter-spring  " 
fevers  of  Italian  writers — there  being  in  this  group  two  distinct 
species,  for  the  quartan  and  tertian  types  respectively ;  whilst 
the  other  includes  the  parasites  of  the  severer  forms — "sestivo- 
autumnal "  fevers,  malignant  or  pernicious  fevers  of  the  tropics, 
or  irregularly  remittent  fevers.  There  is  still  doubt  as  to 
whether  there  are  more  than  one  species  in  this  latter  group. 
Formerly  Italian  writers  distinguished  (1)  a  quotidian ;  (2)  a 
non-pi gmen ted  quotidian ;  and  (3)  a  malignant  tertian  parasite, 
34 


530  MALARIAL   FEVER 

though  the  morphological  differences  described  we're  slight. 
Further  observations  have,  however,  thrown  doubt  on  this  dis- 
tinction, and  the  evidence  rather  goes  to  show  that  there  is  a 
single  species.  Opinion  also  varies  as  to  the  cycle  of  this 
parasite  ;  according  to  some  observers  it  is  twenty-four  hours, 
according  to  others  forty-eight  hours,  though  it  is  generally 
admitted  that  variations  occur.  The  fever  is  often  of  an 
irregular  type  and  multiple  infection  is  probably  common. 
Although  the  question  cannot  be  considered  as  finally  settled,  we 
shall  speak  of  three  species  of  human  parasites.  The  zoological 
position  may  be  shown  by  the  following  scheme,  generally 
followed  by  English  writers,  the  terminology  being  chiefly  that 
of  Grassi  and  Feletti : — 

Family  :  H^MAMCEBID^;  (Wasielewski). 

Genus  I.  Haemamoeba.  The  mature  gametes  resemble  in  form  the 
schizonts  before  segmentation  has  occurred. 

Species  1.  Hceniamceba  DanilewsM  or  halteridium. 
Parasite  of  pigeons,  crows,  etc. 

Species  2.   Hccmamceba,  relicta  or  proteosoina. 
Parasite  of  sparrows,  larks,  etc. 

Species  3.  ffcemamosba  malarice. 

Parasite  of  quartan  fever  of  man. 

Species  4.  Hcemamoeba  vivax. 

Parasite  of  tertian  fever  of  man. 

Genus  II.  Haemomenas.  The  gametocytes  have  a  special  crescentic 
form. 

Species  :  Hcemomenas  prcecox. 

Parasiteof  malignant  orrestivo-autumnalfeverof  man. 

In  addition  there  are  other  species  belonging  to  the  same 
family  of  blood  parasites,  which  infect  frogs,  lizards,  bats,  etc., 
especially  in  malarial  regions. 

We  shall  now  give  the  chief  distinctive  characters  of  the 
three  human  parasites. 

1.  Parasite  of  Quartan  Fever. — The  cycle  of  development  in 
man  is  seventy-two  hours,  and  produces  pyrexia  every  third  day  ; 
double  or  triple  infection  may,  however,  occur.  In  fresh  speci- 
mens of  blood  the  outline  is  more  distinct  than  that  of  the  tertian 
parasite,  and  amceboid  movement  is  less  marked.  Only  the 
smaller  forms  show  movement,  and  this  is  not  of  active  character. 
The  infected  red  corpuscles  do  not  become  altered  in  size  or 
appearance,  and  the  pigment  within  the  parasite  is  in  the  form 
of  coarse  granules,  of  dark  brown  or  almost  black  colour.  The 


VARIETIES   OF   THE   MALARIAL   PARASITE     531 

fully  developed  schizont  has  a  "daisy-head"  appearance, 
dividing  by  regular  radial  segmentation  into  six  to  twelve 
merozoites  which,  on  becoming  free,  are  rounded  in  form. 

2.  The  Parasite  of  Mild  Tertian  Fever. — The  cycle  of  develop- 
ment is  completed  in  forty-eight  hours,  though  a  quotidian  type 
of  fever  may  be  produced  by  double  infection.     The  amoebulae 
have  a  less  refractile  margin  than  in  the  quartan  type,  and  are 
thus  less  easily  distinguished  in  the  fresh  blood  ;  the  amoeboid 
movements  are,  however,  much  more  active,  while  longer  and 
more  slender  processes  are  given  off.     The  infected  corpuscles 
become  swollen  and  pale,  and  may  show  deeply  stained  points 
by  the  Romanowsky    method — "  Schiiffner's    dots."     The   pig- 
ment within  the  parasite  is  fine  and  of  yellowish-brown  tint. 
The  mature  schizont  is  rather  larger  than  in  the  quartan,  has 
a  rosette  appearance,  and  gives  rise  to  fifteen  to  twenty  merozoites, 
though  sometimes  even  more  occur ;  these  have  a  somewhat  oval 
shape. 

In  both  the  quartan  and  tertian  fevers  all  the  stages  of 
development  can  be  readily  observed  in  the  peripheral  blood. 

3.  The  Parasite  of  Malignant  or  JEstivo-autumnal  Fever,  or 
Tropical  Malaria. — The  cycle  in  the  human  subject  probably 
occupies  forty-eight  hours,  though  this  cannot  be  definitely  stated 
to  be  always  the  case  (vide  supra).     The  amoabulse  in  the  red 
corpuscles  are  of  small  size,  and  their  amoeboid  movements  are 
very  active ;  they  often,  however,  pass  into  the  quiescent  ring 
form   (Fig.    160).     The  pigment  granules,  even  in  the  larger 
forms,   are  few   in    number   and   very   fine;   the   infected   red 
corpuscles  have  a  tendency  to  shrivel  and  assume  a  deeper  or 
coppery  tint.     The  fully  developed  schizont  occupies  less  than 
half  the  red  corpuscle,  and  gives  rise  to  usually  from  six  to 
twelve  merozoites,  somewhat  irregularly  arranged  and  of  minute 
size.     Schizogony  takes  place  almost  exclusively  in  the  internal 
organs,  spleen,  'etc.,  so  that,  as  a  rule,  no  sporocytes  can  be 
found  in  the  blood  taken  in  the  usual  way.     The  proportion 
of  red  corpuscles  infected  by  the  amoebulae  is  also  much  larger 
in  the  internal  organs.     The  gametes  have  the  crescentic  form, 
as  already  described. 

Cases  of  infection  with  the  malignant  parasite  sometimes 
assume  a  pernicious  character,  and  then  the  number  of  organisms 
in  the  interior  of  the  body  may  be  enormous.  In  certain  fatal 
cases  with  coma  the  cerebral  capillaries  appear  to  be  almost 
filled  with  them,  many  parasites  being  in  process  of  sporulation  ; 
and  in  so-called  algid  cases,  characterised  by  great  collapse,  a 
similar  condition  has  been  found  in  the  capillaries  of  the 


532  MALARIAL   FEVER 

omentum  and  intestines.  The  process  of  blood  destruction, 
present  in  all  malarial  fevers,  reaches  its  maximum  in  the 
malignant  class,  and  the  brown  or  black  pigment  elaborated  by 
the  parasites — in  part  after  being  taken  up  by  leucocytes,  chiefly 
of  the  mononuclear  class — becomes  deposited  in  various  organs, 
spleen,  liver,  brain,  etc.,  especially  in  the  endothelium  of 
vessels  and  the  perivascular  lymphatics.  In  the  severer  forms 
also  brownish-yellow  pigment  is  apparently  derived  from  liberated 
haemoglobin,  and  accumulates  in  various  parts,  especially  in  the 
liver  cells  ;  most  of  this  latter  gives  the  reaction  of  hsemosiderin. 
General  Considerations. — The  development  of  the  malarial 
parasites  in  the  mosquito  and  infection  of  the  human  subject 
through  the  bites  of  this  insect,  have,  by  the  work  of  Ross  and 
others,  as  detailed  above,  become  established  scientific  facts. 
These  facts,  moreover,  point  to  certain  definite  methods  of  pre- 
vention of  infection,  which  have  to  a  certain  extent  already  been 
practically  tested.  The  extensive  observations  recently  carried 
out  go  to  show  that  all  the  mosquitoes  which  act  as  hosts  of  the 
parasite  belong  to  the  genus  anopheles;  of  these  there  are  a 
large  number  of  species,  and  in  at  least  eight  or  nine  the 
parasite  has  been  found.  Some  of  these  anopheles  occur  in 
England,  especially  in  regions  where  malaria  formerly  prevailed. 
The  opportunity  for  infection  from  cases  of  malaria  returning 
from  the  tropics  to  this  country  thus  exists,  and  such  infection 
has  occurred.  The  breeding-places  of  the  insects  are  chiefly  in 
stagnant  pools  and  other  collections  of  standing  water,  and 
accordingly  the  removal,  where  practicable,  by  drainage  of 
such  collections  in  the  vicinity  of  centres  of  population,  and 
the  killing  of  the  larvae  by  petroleum  sprinkled  on  the  water, 
have  constituted  one  of  the  most  important  measures.  This 
procedure  has  been  carried  out  in  various  places  with  marked 
success.  Another  measure  is  the  protection  against  mosquito 
bites  by  netting,  it  being  fortunately  the  habit  of  the  anopheles 
to  rarely  become  active  before  sundown.  The  experiments  of 
Sambon  and  Low  in  the  Campagna  proved  that  individuals 
using  these  means  of  protection  may  live  in  a  highly  malarial 
district  without  becoming  infected.  The  administration  of 
quinine  to  persons  living  in  highly  malarial  regions,  in  order  to 
prevent  infection,  has  also  been  recommended  and  carried  out. 
In  the  tropics  the  natives  in  large  proportion  suffer  from  malarial 
infection,  and  one  would  accordingly  expect  that  infection  of  the 
mosquitoes  in  the  neighbourhood  of  native  settlements  will  be 
common.  This  has  been  found  to  be  actually  the  case,  and  it 
has  accordingly  been  suggested  that  the  dwellings  of  whites 


THE   PATHOLOGY  -OF   MALARIA  533 

should  as  far  as  possible  be  at  some  distance  from  the  native 
centres  of  population. 

So  far  as  is  known  none  of  the  lower  animals  have  been  found 
to  take  the  place  of  man  as  intermediate  host  to  the  parasite  of 
malaria,  but  the  possibility  of  such  being  the  case  cannot  be  as 
yet  definitely  excluded.  On  the  death  of  infected  mosquitoes 
the  exotospores  or  sporozoites  will  become  set  free,  and  therefore 
theoretically  there  is  a  possibility  that  they  may  enter  the 
human  subject  by  inhalation  or  by  some  other  means.  We  have 
no  facts,  however,  to  show  that  this  really  occurs,  and  the 
evidence  already  obtained  establishes  the  bites  of  mosquitoes  as 
the  most  important  if  not  the  only  mode  of  infection. 

It  may  also  be  mentioned  as  a  scientific  fact  of  some  interest, 
though  not  bearing  on  the  natural  modes  of  infection,  that  the 
disease  can  also  be  communicated  from  one  person  to  another  by 
injecting  the  blood  containing  the  pa.rasites.  Several  experi- 
ments of  this  kind  have  been  performed  (usually  about  ^  to  1  c.c. 
of  blood  has  been  used),  and  the  result  is  more  certain  in 
intravenous  than  in  subcutaneous  injection.  In  such  cases  there 
is  an  incubation  period,  usually  of  from  seven  to  fourteen  days, 
after  which  the  fever  occurs;  the  same  type  of  fever  is  re- 
produced as  was  present  in  the  patient  from  whom  the  blood 
was  taken. 

The  Pathology  of  Malaria. — While  much  work  has  been 
done  on  the  malarial  parasite,  relatively  less  attention  has  been 
directed  to  the  processes  by  which  it  produces  its  pathogenic 
effects.  It  may  be  said  that  the  organisms  are  not  always 
equally  prevalent  in  the  circulating  blood,  and  probably  at 
certain  stages  tend  to  be  confined  in  the  solid  organs ;  thus  they 
may  be  scanty  at  the  height  of  the  paroxysm.  Some  of  the 
pathogenic  effects  are  probably  associated  with  particular  stages 
in  the  life  cycle.  Thus  the  pyrexia  occurs  when  the  stage  of 
sporulation  is  actively  in  progress.  No  opinion  can  be  stated, 
however,  as  to  the  cause  of  the  fever, — whether  it  is  due  to  a 
toxic  process  or  to  general  disturbance  of  metabolism.  We  can 
better  explain  the  anaemia  which  is  so  pronounced  in  cases  where 
the  disease  is  of  long  standing,  and  which  is  due  to  the  actual 
destruction  of  red  blood  corpuscles.  The  parasite  in  its  sojourn 
in  these  cells  absorbs  their  pigment  and  thus  destroys  their 
function ;  this  is  further  evidenced  by  the  activity  displayed  by 
the  red  marrow  in  its  attempts  to  make  good  the  loss  sustained 
by  the  blood.  One  of  the  most  interesting  events  in  malaria, 
and  one  that  links  it  with  bacterial  infections,  is  the  reaction  of 
the  colourless  cells  of  the  blood.  It  has  been  shown  that  during 


534  MALARIAL   FEVER 

the  apyrexial  stages  the  total  number  of  leucocytes  may  be 
diminished  but  that  there  is  always  an  increase  of  the  mono- 
nuclear  cells,  these  frequently  numbering  20  per  cent  of  the 
whole,  and  sometimes  even  outnumbering  the  polymorphs. 
This  is  such  an  important  feature  that  in  cases  where  the 
parasites  themselves  cannot  be  demonstrated  in  the  blood,  the 
mononuclear  reaction,  along  with  the  presence  of  pigment  in  the 
mononuclear  cells  (due  to  phagocytosis  of  pigmented  parasites), 
has  been  taken  as  evidence  that  the  case  is  really  one  of  malaria. 
The  mononuclear  reaction  is  specially  interesting  from  the  fact 
that  in  other  protozoal  diseases  an  activity  of  the  same  elements 
has  been  observed. 

The  question  of  the  possibility  of  immunity  to  malaria  being 
developed  naturally  arises,  and  this  is  specially  interesting  in 
the  light  of  the  leucocytic  reaction  which  we  have  seen  must  be 
looked  on  as  an  element  in  immunity  against  bacterial  infection. 
With  regard  to  Europeans  developing  immunity  it  is  difficult  to 
speak.  In  such  a  malaria-stricken  region  as  the  West  Coast  of 
Africa  the  death-rate  in  residents  of  more  than  four  years' 
standing  is  less  than  in  the  previous  years,  but  this  may  be  due 
to  the  survival  of  the  more  resistant  immigrants.  But  there  can 
be  little  doubt  that  malaria  in  the  negro  is  a  much  less  serious 
condition  than  in  the  European.  Koch  from  his  observations  in 
New  Guinea  attributes  this  to  the  infection  of  the  native  children 
leading  to  the  development  of  immunity  in  the  adult  community. 
He  found,  what  has  been  independently  noted  by  Stephens  and 
Christophers  in  West  Africa,  that  the  greater  number  of  the 
children  harboured  malarial  parasites  in  their  blood.  The  wide- 
spread presence  of  parasites  in  children  might  appear  to  preclude 
the  immunity  of  the  adult  being  due  to  survival  of  the  most 
resistant,  but  the  infant  mortality  in  these  regions  may  be  very 
high,  and  such  a  survival  may  be  the  real  explanation.  On  the 
other  hand,  Koch  states  that  while  an  immunity  appears  to  exist 
in  native  adults  in  malarial  districts,  this  is  only  true  of  those 
born  in  the  locality ;  natives  coming  from  neighbouring  non- 
malarial  districts  into  the  malarial  region  being  liable  to  contract 
the  disease.  At  present  it  must  be  held  that  the  facts  available 
do  not  enable  us  to  determine  the  relative  parts  played  by  the 
development  of  artificial  immunity  on  the  one  hand,  and  the 
existence  of  a  natural  immunity  on  the  other,  in  apparent  un- 
susceptibility  to  malaria. 

Our  knowledge  on  the  relationship  of  blackwater  fever  to 
malaria  is  also  in  an  unsatisfactory  condition.  Blackwater  fever 
is  a  condition  often  occurring,  especially  in  Europeans,  in  tropical 


METHODS   OF   EXAMINATION  535 

countries.  It  is  characterised  by  pyrexia,  darkly-coloured  urine, 
— the  colour  being  due  to  altered  haemoglobin  pigment, — delirium 
and  collapse,  frequently  ending  in  coma  and  death.  By  some 
the  condition  has  been  looked  on  as  a  separate  disease,  by  others 
as  the  terminal  stage  of  a  severe  malaria.  With  regard  to  the 
former  view  no  special  parasite  has  yet  been  demonstrated. 
Stephens  sums  up  the  evidence  for  the  second  view  by  saying 
that  malaria,  apart  from  the  occurrence  of  blackwater  fever,  is  a 
relatively  non-fatal  disease,  that  in  the  great  majority  of  cases 
there  is  direct  or  indirect  evidence  of  the  subject  of  the  condition 
having  suffered  from  repeated  attacks  of  malaria,  that  while  in 
all  cases  there  must  be  an  agent  at  work  causing  haemolysis, 
there  is  evidence  that  in  many  cases  there  is  the  possibility  of 
that  agent  being  quinine.  This  last  point  is  of  great  interest. 
It  has  been  shown  that  in  certain  individuals  the  taking  of  this 
drug  is  sometimes  followed  by  hsemoglobinuria.  The  conditions 
under  which  this  occurs  are  unknown,  and  in  the  case  of  black- 
water  patients,  neither  is  the  serum  haemolytic  for  normal 
corpuscles,  nor  do  the  red  corpuscles  seem  to  be  specially 
sensitive  to  haemolysis  by  quinine,  in  fact,  the  latter  do  not 
appreciably  differ  from  ordinary  red  cells.  The  whole  subject 
of  the  pathology  of  the  condition  is  thus  very  obscure. 

Methods  of  Examination. — The  parasites  may  be  studied  by 
examining  the  blood  in  the  fresh  condition,  or  by  permanent 
preparations.  In  the  former  case,  a  slide  and  cover-glass  having 
been  thoroughly  cleaned,  a  small  drop  of  blood  from  the  finger 
or  lobe  of  the  ear  is  caught  by  the  cover-glass,  and  allowed  to 
spread  out  between  it  and  the  slide.  It  ought  to  be  of  such  a 
size  that  only  a  thin  layer  is  formed.  A  ring  of  vaseline  is 
placed  round  the  edge  of  the  cover-glass  to  prevent  evaporation. 
For  satisfactory  examination  an  immersion  lens  is  to  be  preferred. 
The  amoaboid  movements  are  visible  at  the  ordinary  room 
temperature,  though  they  are  more  active  on  a  warm  stage. 
With  an  Abbe  condenser  a  small  aperture  of  the  diaphragm 
should  be  used. 

Permanent  preparations  are  best  made  by  means  of  dried 
films.  A  small  drop  of  blood  is  allowed  to  spread  itself  out 
between  two  cover-glasses,  which  are  separated  by  sliding  the 
one  on  the  other.  The  films  are  then  allowed  to  dry.  A  very 
good  method  is  that  of  Manson,  who  catches  the  drop  of  blood 
on  a  piece  of  gutta-percha  tissue  (a  piece  of  cigarette-paper  also 
does  well),  and  then  makes  a  film  on  a  clean  slide  by  drawing 
the  blood  over  the  surface.  The  dried  films  are  then  fixed  by 
one  of  the  methods  already  given  (p.  87),  or  by  placing  in 


536  MALARIAL   FEVER 

absolute  alcohol  for  five  minutes  (Manson).  The  films  thus 
prepared  and  fixed  may  be  stained  for  two  or  three  minutes  in  a 
saturated  watery  solution  of  methylene  blue  or  in  carbol-thionin- 
blue  (p.  98) ;  the  solutions  must  be  carefully  filtered  (especially 
the  latter),  and  the  films  must  be  washed  well  after  staining. 
They  are  then  dried  and  mounted  in  balsam.  In  the  case  of 
thionin-blue,  sharper  results  are  obtained  by  dehydrating  in 
alcohol  and  clearing  in  xylol  before  mounting.  The  best  results 
are,  however,  obtained  by  one  of  the  Romanowsky  methods  as 
described  on  p.  106. 

The  fact  that  in  many  cases  the  parasites  may  be  few  in 
number  led  Ross  to  devise  a  method  for  making  their  recognition 
more  easy  by  using  blood  films  of  unusual  thickness.  Here 
about  as  much  blood  as  is  used  in  a  haemoglobin  determination 
(20  c.mm.)  is  taken  on  a  slide,  and,  being  spread  out  only  so 
much  as  to  occupy  the  area  of  an  ordinary  cover-glass,  is  allowed 
to  dry.  There  is  then  dropped  on  it  by  means  of  a  glass  rod  a 
little  of  the  watery  eosin  used  in  making  up  the  Romanowsky 
dye  (vide  p.  106).  This  is  allowed  to  act  for  about  a  quarter 
of  an  hour,  and  then  very  gently  washed  off  with  distilled  water. 
The  Romanowsky  methylene-blue  solution  is  then  applied  for  a 
few  seconds  and  also  carefully  washed  off,  and  the  preparation 
dried  and  mounted.  The  haemoglobin  of  the  red  corpuscles  is 
washed  out  by  the  eosin  solution,  and  the  smaller  forms  of  the 
malarial  parasite  stand  out  as  round  circles  containing  the  char- 
acteristic chromatin  dots ;  and  in  consequence  of  the  greater 
number  present  in  an  area  of  unit  size  as  compared  with  an 
ordinary  preparation  their  recognition  is  very  easy.  For  the 
large  forms  of  the  parasite  Ross  has  found  it  useful  to  make 
such  a  film  and,  haemolysing  the  red  cells  with  distilled  water, 
to  examine  it  unstained.  The  presence  of  pigment  in  the  para- 
sites enables  these  to  be  readily  seen. 


APPENDIX  D. 
AMCEBIC  DYSENTERY. 

IN  a  previous  chapter  it  has  been  pointed  out  that  the  term 
"dysentery"  has  been  applied  to  a  number  of  conditions  of 
different  etiology,  and  the  relations  of  bacteria  as  causal  agents 
have  been  there  discussed  (vide  p.  346).  We  shall  here  consider 
that  variety  of  tropical  dysentery  which  is  believed  to  be  due  to 
an  amoeba,  and  hence  often  known  as  amoebic  dysentery. 

Amongst  the  early  researches  on  the  relation  of  organisms  to 
dysentery  probably  the  most  important  are  those  of  Losch,  who 
noted  the  presence  and  described  the  characters  of  amoebae  in 
the  stools  of  a  person  suffering  from  the  disease,  and  considered 
that  they  were  probably  the  causal  agents.  Further  observations 
on  a  more  extended  scale  were  made  by  Kartulis  with  confirma- 
tory results,  this  observer  finding  the  same  organisms  also  in 
liver  abscesses  associated  with  dysentery.  Councilman  and 
Lafleur,  working  in  Baltimore,  showed  that  this  variety  of 
dysentery  can  be  distinguished  from  other  forms,  not  only  by 
the  presence  of  amoebae  but  also  by  its  pathological  anatomy. 
The  intestinal  lesions,  to  which  reference  is  made  below,  are  of  a 
grave  character,  mortality  is  relatively  high,  and  recovery,  when 
it  occurs,  is  protracted  on  account  of  the  extensive  tissue 
changes.  The  subject  was,  however,  complicated  by  the  fact 
that  a  similar  organism — the  amoeba  cofo'— had  been  previously 
found  in  the  intestine  in  normal  conditions  and  in  other 
diseases  than  dysentery  (by  Cunningham  and  Lewis  and  others), 
and  additional  research  confirmed  these  results.  It  may  now  be 
regarded  as  established  that  the  amoeba  of  dysentery  and  the 
common  amoeba  of  the  colon  are  two  distinct  species.  This  has 
especially  been  shown  by  the  researches  of  Schaudinn,  who  has 
given  the  terms  entamoeba  histolytica  and  entamoeba  coli  to  the 
two  organisms. 

Entamoeba  histolytica  as  seen  in  the  dysenteric  stools  occurs 

537 


538 


AMCEBIC   DYSENTERY 


in  the  form  of  rounded,  oval,  or  pear-shaped  cells,  measuring 
12-30  /*.  in  diameter.  When  at  rest  a  somewhat  clear,  highly 
refractile  ectoplasm  and  a  granular  endoplasm  can  be  distin- 
guished, a  feature  which  differentiates  the  organism  from  the 
entamoeba  coli.  The  nucleus  is  rounded,  or  oval,  and  is  seen 
with  difficulty  ;  its  position  is  usually  eccentric  and  is  sometimes 
quite  at  the  margin  of  the  ectoplasm.  In  the  fresh  condition, 
and  especially  when  examined  on  a  warm  stage,  the  organism 
shows  very  active  amoeboid  movements.  The  pseudopodia,  which 
are  quickly  protruded  and  retracted,  are  blunt  and  appear  to  be 
of  tough  consistence  (Fig.  166),  a  property  which  Schaudinn 
considers  of  importance  as  enabling  the  organism  to  penetrate 
the  mucous  membrane,  etc.  The  amoebic  movements  are  often 

14 


FIQ.  166. — Amoebse  of  dysentery. 

a  and  b,  amuebie  as  seen  in  the  fresh  .stools,  showing  blunt  amoeboid  processes  of 
ectoplasm.  The  endoplasm  of  a  shows  a  nucleus,  three  red  corpuscles  and  numerous 
vacuoles  ;  that  of  b,  numerous  red  corpuscles  and  a  few  vacuoles. 

c,  an  amoeba  as  seen  in  a  fixed  film  preparation,  showing  a  small  rounded  nucleus 
(Kruse  and  Pasquale).  xGOO. 

of  an  active  kind  and  locomotion  may  be  fairly  rapid ;  not 
infrequently  red  corpuscles,  bacteria,  cells,  etc.  may  be  seen  in 
the  interior.  The  organism  usually  dies  and  undergoes  disinte- 
gration in  a  comparatively  short  time  after  being  removed  from 
the  body ;  the  stools  ought  therefore  to  be  examined  in  as  fresh 
a  state  as  possible.  Multiplication  takes  place  by  simple  amitotic 
division  and  also  by  budding.  The  entamoeba  coli  is  an  organism 
of  about  the  same  size.  When  at  rest  it  shows  no  differentiation 
into  ectoplasm  and  endoplasm,  and  the  nucleus,  usually  situated 
in  the  centre,  shows  a  highly  refractile  membrane  with  chromatin 
masses  scattered  in  the  interior.  During  amoeboid  movement 
some  delicate  processes  of  ectoplasm  come  into  view. 


DISTRIBUTION   OF   THE   AMCEB^E  539 

Both  organisms  have  now  been  shown  to  pass  into  a  resting 
stage  with  formation  of  cysts,  the  character  and  mode  of  forma- 
tion of  which  are  markedly  different  in  the  two  cases.  The  cyst 
formation  of  the  entamoeba  histolytica  is  specially  seen  when  the 
disease  is  in  process  of  cure  and  the  stools  are  beginning  to  have 
a  less  fluid  character.  In  the  earliest  stage  of  the  change  the 
nuclear  membrane  becomes  broader  and  fades  into  the  proto- 
plasm, whilst  the  chromatin  becomes  dispersed  through  the 
entoplasm  in  the  form  of  small  chromidia.  Buds  then  form 
on  the  surface  and  into  these  some  of  the  chromatin  passes. 
Around  these  buds  concentric  striation  can  be  seen,  and  then 
a  hyaline  cyst  wall  is  formed,  which  is  highly  refractile  in 
character.  The  cyst  then  becomes  separated  from  the  rest  of  the 
cell.  Several  cysts  which  measure  2-7  p  in  diameter  may  be 
formed  from  the  same  amoeba,  and  the  remnant  of  the  cell  under- 
goes disintegration.  These  cysts,  as  will  be  shown  below,  repre- 
sent a  resting  stage  with  high  powers  of  resistance  to  external 
agencies,  and  are  concerned  in  producing  infection  of  another 
subject.  The  cellular  changes  in  the  encysting  of  the  entamceba 
coli  have  also  been  worked  out  by  Schaudinn.  They  are  of 
a  somewhat  complicated  character,  involving  the  formation 
of  reduction  bodies  and  copulation  of  nuclei,  but  the  ultimate 
result  is  the  formation  of  a  fairly  large  cyst,  which  contains 
eight  small  cells.  The  process  of  cyst  formation  accordingly  in 
the  two  organisms  is  of  a  widely  different  character. 

Cultivation. — Various  attempts  have  been  made  to  cultivate 
the  amoeba  of  dysentery,  and  Kartulis  considered  that  he  obtained 
growth  in  straw  infusions.  Recently  Lesage  has  announced  that 
he  has  obtained  cultures  on  plates  of  agar  which  had  been 
washed  in  water  for  eight  days.  Both  the  vegetative  and  the 
cystic  forms  were  used  for  inoculation.  In  some  cases  a  growth 
of  a  colon  bacillus  was  made  on  the  agar  and  afterwards  removed, 
this  procedure  interfering  with  the  development  of  such  bacilli 
present  in  the  material  used  for  inoculation.  The  plates  were 
kept  in  the  sloped  position,  and  the  inoculations  were  made  in 
the  lower  part ;  the  amoebae  moved  to  the  upper  part,  where  they 
were  got  in  pure  condition.  He  succeeded  in  obtaining  cultures 
in  seven  out  of  thirty  cases,  and  in  some  instances  cultivated  the 
organisms  for  more  than  sixty  generations.  The  amoebae  multi- 
plied by  simple  amitotic  division,  and  in  certain  cases  produced 
small  cysts.  These  cysts,  as  described  and  figured  by  him, 
correspond  in  all  important  respects  with  the  changes  observed 
by  Schaudinn  in  dysenteric  stools. 

Distribution  of  the  Amrebae. — As  already  stated,  they  are 


540 


AMCEBIC   DYSENTERY 


usually  found  in  large  numbers  in  the  contents  of  the  large 
intestine,  in  tropical  amoebic  dysentery.  They  also,  however, 
penetrate  into  the  tissues,  where  they  appear  to  exert  a  well- 
marked  action.  In  this  disease  the  lesions  are  chiefly  in  the  large 
intestine,  especially  in  the  rectum  and  at  the  flexures,  though 
they  may  also  be  present  in  the  lower  part  of  the  ileum.  At 
first  there  are  seen  local  swellings  on  the  mucous  surface,  chiefly 
due  to  a  sort  of  inflammatory  gelatinous  oedema  with  little 
leucocytic  infiltration ;  soon,  however,  the  mucous  membrane 
becomes  partially  ulcerated,  more  or  less  extensive  necrosis  of 

the  subjacent  tissues  oc- 
curs, and  gangrenous 
sloughs  result.  The  ulcers 
thus  come  to  have  irregular 
and  overhanging  margins, 
and  the  excavation  below 
is  often  of  wider  extent 
than  the  aperture  in  the 
mucous  membrane.  The 
amoebae  are  found  in  the 
mucous  membrane  when 
ulcers  are  being  formed, 
but  their  most  character- 
istic site  is  beyond  the 
ulcerated  area,  where  they 
may  be  seen  penetrating 
deeply  into  the  submucous, 
and  even  into  the  muscular 
coats.  In  these  positions 
they  may  be  unattended 
by  any  other  organisms, 

and  the  tissues  around  them  show  cedematous  swelling  and  more 
or  less  necrotic  change  without  much  accompanying  cellular 
reaction,  beyond  a  certain  amount  of  swelling  and  proliferation 
of  the  connective-tissue  cells.  This  action  of  the  amoebae  on 
the  tissues  explains  the  character  of  the  ulcers  as  just  de- 
scribed. These  lesions  are  considered  to  be  characteristic  of 
amoebic  dysentery. 

As  a  complication  of  this  form  of  dysentery,  liver  abscesses 
are  of  comparatively  common  occurrence.  They  are  usually 
single  and  of  large  size,  sometimes  there  are  more  than  one,  and 
occasionally  numerous  small  ones  may  be  present.  The  contents 
are  usually  a  thick  pinkish  fluid  of  somewhat  slimy  consistence 
and  are  largely  constituted  by  necrosed  and  liquefied  tissue  with 


FIG.  167. — Section  of  wall  of  liver  abscess, 
showing  an  amoeba  of  spherical  form  with 
vacuolated  protoplasm.  From  a  case 
published  by  Surgeon-Major  D.  G. 
Marshall.  x  1000. 


EXPERIMENTAL   INOCULATION  541 

admixture  of  blood  in  varying  amount.  Microscopic  examination 
shows  chiefly  necrosed  and  granular  cells  and  debris  resulting 
from  their  disintegration,  whereas  ordinary  pus  corpuscles  are 
scanty  or  may  be  practically  absent.  In  such  abscesses  associated 
with  dysentery  the  amoebae  are  usually  to  be  found,  and  not 
infrequently  are  the  only  organisms  present,  no  cultures  of 
bacteria  being  obtainable  by  the  ordinary  methods  (Fig.  167). 
They  are  most  numerous  at  the  spreading  margin,  and  this 
probably  explains  a  fact  pointed  out  by  Manson,  that  examination 
of  the  contents  first  removed  may  give  a  negative  result,  while 
they  may  be  detected  in  the  discharge  a  day  or  two  later.  The 
action  here  on  the  tissues  is  of  an  analogous  nature,  namely,  a 
necrosis  with  softening  and  partial  liquefaction,  attended  by 
little  or  no  suppurative  change.  The  amoebae  have  also  been 
found  in  the  sputum  when  a  liver  abscess  has  ruptured  into  the 
lung,  as  not  very  infrequently  happens.  Kartulis  records  two 
cases  of  brain  abscess  occurring  secondarily  to  dysentery  in 
which  numerous  amoebae  were  present. 

Experimental  Inoculation. — The  anatomical  changes  in 
dysentery,  as  above  described,  gives  strong  presumptive  evidence 
as  to  the  causal  relationship  of  the  amoebae,  and  practically  con- 
clusive evidence  is  afforded  by  animal  experiments.  Dysentery 
occurs  occasionally  in  animals,  but  it  is  of  rare  occurrence.  The 
disease  sometimes  results  in  the  dog  by  experimental  inoculation 
with  dysenteric  material.  Kartulis,  for  example,  records  two 
cases,  in  one  of  which  liver  abscess  was  present.  Cats  are, 
however,  found  to  be  more  susceptible,  especially  young  animals. 
Dysenteric  changes  have  been  produced  in  this  animal  by 
Kartulis,  Kruse  and  Pasquale,  and  others.  The  method  generally 
adopted  is  the  introduction  of  a  small  quantity  of  mucus  from 
a  dysenteric  case  into  the  rectum.  The  resulting  disease  is  of 
an  acute  character,  and  sometimes  leads  to  a  fatal  result.  The 
changes  in  the  -large  intestine  resemble  those  found  in  the 
human  disease,  and  microscopic  examination  shows  the  amoebae 
penetrating  the  wall  of  the  bowel  in  the  characteristic  manner. 
Kruse  and  Pasquale  obtained  corresponding  results  when  the 
material  from  a  liver  abscess,  containing  amoebae  without  any 
other  organisms,  was  injected.  Quincke  and  Roos  obtained  no 
effects  when  the  amoebae  were  administered  by  the  mouth,  but 
they  obtained  a  fatal  result  in  two  out  of  four  cases  when  the 
cyst-like  forms  were  given.  They  also  found  that  the  cysts, 
unlike  the  amoebae,  were  still  present  even  after  the  material 
had  been  kept  for  two  or  three  weeks.  Extremely  important 
confirmatory  evidence  with  regard  to  infection  by  the  cysts  has 


542  AMCEBIC   DYSENTERY 

been  supplied  by  experiments  of  Schaudinn.  Dysenteric  material 
was  obtained  from  China,  and  portions  of  it  which  were  found 
to  contain  the  cysts  were  thoroughly  dried.  Some  of  this 
material  was  given  with  food  to  cats  by  the  mouth,  and  typical 
dysentery  resulted,  the  amoebae  being  found  in  the  stools.  No 
results  follow  when  the  material  ingested  merely  contains  the 
vegetative  form  of  the  organism,  as  it  is  readily  destroyed  in  the 
contents  of  the  stomach. 

Investigations  with  regard  to  entamoeba  coli  show  that  it  is 
a  harmless  organism  and  that  it  is  frequently  present  in  the 
intestines  of  healthy  individuals.  Schaudinn  found  that  in  East 
Prussia  as  many  as  50  per  cent  of  the  population  were  infected 
with  it.  The  administration  of  the  amoebae,  or  of  the  cysts  by 
the  methods  mentioned  above,  produces  no  result  in  animals. 
It  has,  however,  been  shown  that  when  the  eight-celled  cysts 
are  swallowed  by  persons  who  are  free  from  the  parasite,  the 
entamceba  coli  appears  in  the  large  intestine  in  a  comparatively 
short  period  of  time.  It  accordingly  appears  that  in  the  case  of 
both  organisms  it  is  the  cysts  alone  which  give  rise  to  infection. 

From  the  above  facts,  all  of  which  have  received  ample 
confirmation,  there  can  be  no  doubt  that  the  amoeba  described 
is  the  cause  of  the  form  of  dysentery  with  which  it  is  associated. 
We  are  still  ignorant  whether  the  organism  has  any  life  history 
outside  the  body,  but  it  has  been  shown  that  the  cysts  have 
high  powers  of  endurance  and  almost  certainly  form  the  means 
of  infection  when  they  are  swallowed  in  drinking-water  or  in 
food.  It  is  also  of  importance  to  note  that  the  serum  of  patients 
suffering  from  amoebic  dysentery  gives  no  agglutinating  reaction 
with  Shiga's  bacillus  of  dysentery  (vide  p.  348). 

Methods  of  Examination. — The  faeces  in  a  case  of  suspected 
dysentery  ought  to  be  examined  microscopically  as  soon  as 
possible  after  being  passed,  as  the  amoebae  disappear  rapidly, 
especially  when  the  reaction  becomes  acid.  A  drop  is  placed 
on  a  slide  without  the  addition  of  any  reagent,  a  cover-glass  is 
placed  over  it  but  not  pressed  down,  and  the  preparation  is 
examined  in  the  ordinary  way  or  on  a  hot  stage,  preferably  by 
the  latter  method,  as  the  movements  of  the  amoebae  become  more 
active,  and  it  is  difficult  to  recognise  them  when  they  are  at  rest. 
Hanging-drop  preparations  may  also  be  made  by  the  methods 
described.  Dried  films  are  not  suitable,  as  in  the  preparation  of 
these  the  amoebae  become  broken  down ;  but  wet  films  may  be 
fixed  with  corrosive  sublimate  or  other  fixative  (vide  p.  88).  In 
sections  of  tissue  the  amoebae  may  be  stained  by  methylene-blue, 
by  safranin,  by  haematoxylin  and  eosin,  etc.  Benda's  method  of 


METHODS   OF   EXAMINATION  543 

staining  with  safranin  and  light-green  is  also  a  very  suitable  one. 
Sections  are  stained  for  several  hours  in  a  saturated  solution 
of  safranin  in  aniline  oil  water  (p.  98),  they  are  then  washed 
in  water  and  decolorised  in  a  J  per  cent  solution  of  light-green 
in  alcohol  till  most  of  the  safranin  is  discharged,  the  nuclei, 
however,  remaining  deeply  stained.  In  this  method  the  nuclei 
of  the  amoebae  are  coloured  red  (like  these  of  the  tissue  cells), 
the  protoplasm  being  of  a  purplish  tint. 


APPENDIX   E. 

TRYPANOSOMIASIS— KALA-AZAR—PIROPLASMOSIS. 

THE  PATHOGENIC  TEYPANOSOMES. 

THE  trypanosomata  are  protozoal  organisms  belonging  to  the 
sub-class  Flagellata,  and  during  the  last  decade  several  members 
of  the  genus  have  come  to  be  recognised  as  living  in  the  blood 
and  tissues  in  various  animals  and  as  causing  important  disease 
conditions.  As  long  ago  as  1878  the  Trypanosoma  Lewisi  was 
observed  infesting  the  blood  of  rats,  and  it  has  been  found  to  be 
sometimes  capable  of  causing  death.  Other  diseases  in  which 
similar  organisms  have  been  found  are  Surra,  which  occurs  in 
cattle,  horses,  and  camels  in  India,  and  which  is  associated  with 
the  Tr.  Evansi  ;  Dourine,  a  condition  affecting  horses  in  especially 
the  Mediterranean  littoral  (Tr.  equiperdvm  or  Rougeti)  ;  Mai  de 
Caderas,  a  disease  of  South  American  horses  (Tr.  equinum  or 
Elmassiani) ;  Tse-tse  Fly  Disease  or  Nagana,  affecting  horses 
and  herbivora  in  South  Africa  (Tr.  JBrucei) ;  trypanosomiasis  of 
African  cattle  (Tr.  Theileri) ;  and — most  important  from  the 
human  standpoint — the  trypanosomiasis  and  sleeping  sickness 
of  West  and  Central  Africa  associated  with  the  Tr.  gambiense 
and  Tr.  ugandense,  which  are  now  believed  to  be  the  same 
organism.  These  diseases  present  many  general  resemblances  to 
one  another.  They  tend  to  be  characterised  by  wasting,  cachexia, 
anaemia,  fever  often  of  an  intermittent  type  and  irregular  cedemas, 
and  often  lead  to  a  fatal  result.  In  many  cases  the  infective 
agent  is  conveyed  from  a  diseased  to  a  healthy  animal  by  the 
agency  of  blood-sucking  insects. 

General  Morphology  of  the  Trypanosomata. — If  a  drop  of 
blood  containing  trypanosomes  be  examined,  the  organism  will 
be  seen  to  be  a  fusiform  mass  of  protoplasm  which  at  one  end 
passes  into  a  pointed  flagellum.  In  the  living  condition  the 
trypanosome  is  usually  actively  motile  by  an  undulatory  move- 

544 


THE   PATHOGENIC   TRYPANOSOMES  545 

ment  of  its  protoplasm  and  a  lashing  of  the  flagellum.  The  size 
varies,  but  those  mentioned  above  are  about  30  /x  long  and  about 
1  '5  to  3  //.  broad.  From  the  fact  that  in  progression  the  flagellum 
is  in  front,  the  flagellated  end  is  denominated  the  anterior  end 
of  the  organism.  It  is  stated  that  the  method  of  examining 
the  fresh  blood  merely  allowed  to  spread  itself  out  in  a 
fairly  large  drop  beneath  a  cover-glass  is  more  likely  to  reveal 
the  presence  of  trypanosomes,  if  these  are  present  in  small 
numbers,  than  is  the  examination  of  stained  specimens;  but  the 
minuter  structure  of  the  organisms  can  best  be  studied  in  dried 
preparations  stained  by  Romanowsky  dyes  such  as  those  of 
Leishman  or  Giemsa. 

For  staining  trypanosomata  (or  the  Leishman-Donovan  bodies)  in 
sections  so  as  to  bring  out  the  chromatin  structures,  Leishman  recom- 
mends the  following  method: — Sections  of  5  ft  thickness  are  made  and 
carefully  fixed  on  slides.  The  paraffin  is  very  thoroughly  removed  by 
melting  it  before  applying  the  first  xylol,  and  then  washing  with 
alternate  baths  of  alcohol  and  xylol  three  or  four  times.  The  last 
alcohol  is  thoroughly  washed  off  by  distilled  water,  and  the  excess  of 
water  is  removed  with  cigarette  paper.  A  drop  of  fresh  blood  serum  is 
then  placed  on  the  preparation  and  allowed  to  soak  in  for  five  minutes. 
The  excess  is  removed  by  blotting,  and  the  remainder  is  allowed  to 
dry  on  the  section,  which  is  now  treated  with  a  mixture  of  two  parts 
of  Leishman's  stain  and  three  of  distilled  water,  and  placed  in  a  Petri 
dish  for  1  to  1^  hours.  The  preparation  is  very  deeply  stained,  the 
nuclei  being  almost  black  and  decolorisation  and  differentiation  are 
effected  by  alternately  applying  the  acetic  acid  and  caustic  soda  solutions 
(commencing  with  the  acid)  used  in  the  application  of  the  stain  to 
ordinary  histological  sections  (v.  p.  106),  the  effects  .being  carefully 
watched  with  a  low  power.  The  essential  part  of  the  method  is  the 
application  of  the  blood  serum,  though  what  effect  this  has  is  not 
known  ;  Leishman  suggests  that  it  restores  the  normal  alkalinity  of  the 
tissue. 

In  preparations  stained  by  the  above  methods  the  protoplasm 
of  trypanosomata  stains  blue,  and  in  some  species  some  parts  are 
more  intensely  coloured  than  others.  Sometimes  it  contains 
violet-coloured  granules  (chromatin  granules),  and  occasionally 
there  appears  in  it  slight  longitudinal  striation.  Two  bodies  are 
always  present  in  the  protoplasm.  Usually  near  the  middle  there 
is  an  oval  granular  body  staining  purple, — the  nucleus  or  macro- 
nucleus, — and  towards  the  posterior  end  is  a  minute  intensely 
stained  purple  granule  known  as  the  micronucleus  or  centrosome 
(that  this  body  represents  the  centrosome  is  strongly  held  by 
Laveran  from  the  analogy  of  appearances  in  certain  spermatozoa 
which  closely  resemble  trypanosomes  in  structure).  This  micro- 
nucleus  is  often  surrounded  by  an  unstained  halo,  and  in  its 
neighbourhood,  in  certain  species,  a  vacuole  has  been  described 
35 


546  TRYPANOSOMIASIS 

as  existing ;  this  has  been  considered  by  some  to  be  analogous 
to  the  contractile  vacuole  present  in  many  protozoa,  and  its 
shape  and  position  have  been  made  the  basis  of  specific  dis- 
tinctions ;  Laveran,  however,  thinks  it  is  an  artefact.  From 
the  micronucleus  or  from  its  neighbourhood  there  arises  an 
important  structure  in  the  trypanosome,  —  the  undulatory 
membrane.  This  is  of  varying  breadth,  has  a  sharp  undulating 
free  margin,  and  surmounts  the  protoplasm  of  the  organism 
like  a  cock's  comb;  it  narrows  towards  the  anterior  end 
where  it  passes  into  the  flagellum.  Motion  is  chiefly  effected  by 
the  undulations  of  this  membrane  and  of  the  flagellum.  The 
latter  is  continuous  with  the  protoplasm  of  the  body  of  the 
organism ;  it  stains  uniformly  like  it,  except  the  free  edge  which 
has  the  reddish  hue  of  the  chromatin.  In  different  species 
of  trypanosomes  variations  occur  in  shape,  in  length,  in  breadth, 
in  the  position  of  the  micronucleus  (and  therefore  in  the 
length  of  the  undulating  membrane),  in  the  breadth  of  the 
membrane,  in  the  length  of  the  free  part  of  the  flagellum,  in  the 
shape  of  the  posterior  end,  which  is  sometimes  blunt,  sometimes 
sharp,  and  in  the  presence  or  absence  of  free  chromatin  granules 
in  the  protoplasm. 

Multiplication  in  the  body  fluids  ordinarily  occurs  by 
longitudinal,  amitotic  division  (see  Fig.  168).  First  of  all  the 
micronucleus  divides,  sometimes  transversely,  sometimes  longitud- 
inally, then  the  nucleus  and  undulating  membrane,  and  lastly 
the  protoplasm.  In  some  species  the  root  of  the  flagellum  only 
divides  so  that  in  the  young  trypanosomes  the  flagellum  is 
short  and  subsequently  increases  in  length  (Tr.  Lewisi) ;  usually 
the  whole  flagellum  takes  part  in  the  general  splitting  of  the 
organism. 

In  the  cases  of  several  of  the  trypanosomata  it  has  been 
found  possible  to  cultivate  them  outside  the  body,  the  chief 
work  here  having  been  done  by  Novy  and  MacNeal,  who  have 
succeeded  with  the  Tr.  Lewisi,  Tr.  Evansi,  and  Tr.  Brucei. 
The  most  suitable  medium  is  made  as  follows  : — 

125  grammes  rabbit  or  ox  flesh  is  treated  with  1000  c.c.  distilled 
water,  as  in  making  ordinary  bouillon,  and  there  are  added  to  the 
meat  extract  20  gr.  Witte's  peptone,  5  gr.  sodium  chloride,  20  gr. 
agar,  and  10  c.c.  normal  sodium  carbonate.  The  medium  is  placed  in 
tubes  and  sterilised  in  the  autoclave  at  110°  C.  for  thirty  minutes.  It  is 
cooled  to  50°  C. ,  and  there  is  added  to  the  medium  in  each  tube  twice  its 
volume  of  defibrinated  rabbit  blood,  which  has  been  prepared  with  all 
aseptic  precautions  ;  the  tubes  are  allowed  to  set  in  the  inclined  position. 
In  inoculating  such  tubes  they  are  placed  in  an  upright  position  for  a 
few  minutes  and  then  the  infective  material  is  introduced. 


THE   PATHOGENIC   TRYPANOSOMES  547 

Multiplication  goes  on  readily  on  such  a  medium,  the 
organisms  dividing  longitudinally  as  in  the  tissues.  Sometimes 
very  small  forms  result,  and  often  these  are  found  in  rosettes 
which  are  formed  by  a  number  of  individuals  arranging  them- 
selves in  a  circle  with  the  flagella  directed  towards  the  centre 
of  the  agglomeration.  By  repeated  sub-cultures  several  of  the 
trypanosomata  named  have  been  kept  alive  for  more  than  a  year, 
and  when  re-introduced  into  appropriate  hosts  have  been  found 
not  to  have  lost  their  infective  properties. 

Within  recent  years  considerable  attention  has  been  directed 
to  the  question  of  whether  in  the  trypanosomes  a  sexual 
cycle  occurs.  It  cannot  be  said  that  the  existence  of  such  has 
been  definitely  established,  and  we  shall  merely  give  a  short 
account  of  certain  views  which  have  been  advanced.  The 
starting  point  lies  in  the  slight  differences  in  form  which  have 
been  observed  in  the  organisms  in  the  body  fluids  of  the 
vertebrate  hosts.  Such  differences  have  been  described  in 
Tr.  Lewisi  and  Tr.  Brucei  by  Prowazek,  and  in  Tr.  ugandense 
by  Minchin,  and  have  been  made  the  basis  of  a  classification  into 
three  types  which  are  looked  on  as  representing  male,  female,  and 
indifferent  individuals.  The  male  type  is  rather  slender  both  in 
body  and  in  nucleus,  the  free  part  of  the  flagellum  is  longer  than 
the  body  and  the  protoplasm  is  free  from  granules ;  the  female 
is  broader,  its  nucleus  is  larger  and  rounder,  the  undulating 
membrane  narrower,  the  free  part  of  the  flagellum  is  shorter 
than  the  body,  and  the  protoplasm  contains  many  chromatin 
granules,  which  are  looked  upon  as  reserve  food  material.  The 
indifferent  individuals  present  intermediate  characters.  All 
multiply  by  fission  as  described,  and  the  indifferent  individuals 
can  on  occasion  become  differentiated  into  male  or  female 
forms.  The  females  are  the  most  hardy,  and  next  come  the 
indifferent  individuals.  If  all  but  the  females  die  out,  these 
can  undergo  .parthenogenesis,  and  representatives  of  all  three 
types  can  be  again  reproduced.  The  sexual  cycle  is  represented 
as  occurring  in  the  invertebrate  host.  In  Tr.  Lewisi,  according 
to  Prowazek,  this  is  found  in  the  rat  louse,  hcematopinus 
spinulosus.  When  this  insect  sucks  the  blood  of  an  infected 
rat,  copulation  occurs  by  the  male  trypanosome  entering  the 
female  near  the  micronucleus  and  the  various  parts  of  the  two 
individuals  becoming  fused.  A  non-flagellated  ookinete  results, 
which,  passing  through  a  spindle-shaped  gregarine-like  stage, 
can  develop  into  a  trypanosome  in  the  stomach  of  the  louse. 
A  resting-stage  in  an  immature  trypanosome -like  form  is 
described  as  occurring  between  or  attached  to  the  intestinal 


548  TRYPANOSOMIASIS 

epithelium,  and  the  parasite  is  supposed  to  reach  the  body 
cavity,  and  ultimately  the  pharynx  of  the  insect,  and  thus  to 
find  the  opportunity  for  passing  into  the  body  of  a  fresh  host. 

A  still  further  development  of  the  views  held  as  to  the  life-history  of 
the  trypanosomes  is  found  in  the  work  of  Schaudinn,  who  investigated 
the  trypanosoma  noduce  found  in  the  owl  (athene  noctua),  and  which  is 
carried  from  bird  to  bird  by  the  common  mosquito  (culex  pipiens).  In 
the  blood  of  the  owl  is  a  halteridium  hsemamoeba  showing  pigmented 
male  and  female  forms,  closely  corresponding  to  those  observed  by 
Macallum  in  the  crow.  These,  according  to  Schaudinn,  on  reaching  the 
mosquito's  stomach  undergo  ordinary  changes — the  microgametocyte 
develops  microgametes,  one  of  which  fertilises  the  macrogamete.  An 
oval  motile  ookinete  results,  and  in  the  formation  of  its  nucleus  from 
the  male  and  female]  elements  a  reduction  of  chromosomes  takes  place, 
while  the  superfluous  nuclear  structures  along  with  the  pigment  are  cast 
out  of  the  cell.  In  these  ookinetes  a  differentiation  into  male,  female, 
and  indifferent  forms  can  be  recognised,  but  the  important  new  departure 
is  that  each  can  go  on  to  develop  into  a  trypanosome.  In  the  indifferent 
ookinete  a  portion  of  the  new  nucleus  breaks  off  by  a  sort  of  heteropolar 
division,  and  the  broken-off  part  becomes  the  micronucleus  or  blepharo- 
plast  of  the  trypanosome  and  gives  origin  to  the  undulatory  membrane. 
Longitudinal  division  of  these  forms  in  the  mosquito's  intestine  may 
occur,  and  here  it  may  be  said  that  Schaudinn  differs  from  other  observers 
in  holding  that  on  division  the  membrane  does  not  split,  but  that  one  of 
the  individuals  gets  its  membrane  by  this  being  laid  down  along  the  root 
of  that  already  in  existence.  Further,  a  resting-stage  of  the  trypanosome 
may  occur,  in  which  it  becomes  attached  to  the  intestinal  epithelium, 
and  losing  more  or  less  its  flagellate  form,  may  resemble  a  gregarine. 
The  female  ookinete  is  plumper  and  contains  more  chromatin  granules 
in  its  protoplasm  than  the  male  and  indifferent  forms,  gives  rise  to  a 
trypanosome  after  a  more  complicated  division  of  its  nucleus,  is  less 
motile,  does  not  reproduce  itself  by  longitudinal  fission,  soon  attaches 
itself  to  the  intestinal  epithelium,  is  in  virtue  of  the  reserve  material 
in  its  protoplasm  much  more  resistant  than  the  male  or  indifferent 
forms  ;  when  these  die  out,  as  they  do  when,  for  instance,  the  insect  is 
starved,  it  reproduces  all  three  forms  by  a  process  of  parthenogenesis. 
In  the  female  ookinete  the  smaller  nucleus  which  goes  to  form  the 
blepharoplast  of  the  indifferent  trypanosome  divides  into  eight  small 
nuclei,  all  of  which  perish,  and  the  blepharoplast  and  membrane  are 
formed  by  a  fresh  division  in  the  large  nucleus  remaining.  In  the  male 
ookinete,  which  differs  from  the  female  in  the  clearness  of  its  protoplasm, 
a  similar  heteropolar  mitosis  takes  place,  and  again  eight  small  nuclei 
are  produced.  These  evidently  represent  the  essentially  male  element, 
for  they  persist,  and  each,  appropriating  to  itself  a  portion  of  the 
cellular  protoplasm,  detaches  itself  so  that  eight  small  trypanosomes  are 
budded  off  from  the  ookinete.  This  male  ookinete  Schaudinn  holds  to 
be  homologous  with  the  microgametocyte  occurring  in  the  blood  of  the 
owl,  and  the  small  trypanosomes  are  similarly  homologous  with  the 
microgametes  formed  when  the  blood  of  the  host  reaches  the  stomach 
of  the  mosquito.  These  small  trypanosomes  and  the  male  trypano- 
somes readily  die,  probably  because,  by  a  reduction  process  in  their 
genesis,  the  assimilative  powers  of  the  larger  nucleus  have  been 
diminished.  In  degenerating  they  often  are  found  in  the  intestinal 


THE   PATHOGENIC   TRYPANOSOMES          549 

tract  of  the  mosquito  arranged  in  rosettes,  with  sometimes  the  anterior 
ends,  sometimes  the  posterior  ends,  directed  towards  the  centre  of  the 
rosette.  The  next  stage  of  development  takes  place  when  the  parasites 
by  the  mosquito's  bite  reach  the  blood  of  the  vertebrate  host,  and  it  is 
in  connection  with  this  stage  that  Schaudinn's  observations  are  very 
far-reaching.  The  indifferent  forms  by  this  time,  by  repeated  longi- 
tudinal division,  have  become  small.  In  the  owl  they  attach  them- 
selves to  red  blood  corpuscles,  which  they  penetrate,  and,  assuming  a 
halteridiuin  form,  grow  in  size  for  twenty-four  hours  ;  they  then  leave 
the  cells,  elongate,  again  assume  the  flagellate  form,  move  freely  in  the 
blood  till  the  next  night,  when  they  again  enter  fresh  cells.  This  cycle 
is  repeated  for  six  nights,  the  organism  gaining  in  size  with  each  sojourn 
in  a  corpuscle.  When  the  full  size  is  thus  attained,  repeated  longi- 
tudinal division  occurs,  and  when  the  smallest  forms  are  again  reached 
the  intra- cellular/ cycle  recommences.  The  largest  female  forms  prob- 
ably cannot  pass  through  the  mosquito's  proboscis,  but  when  small 
forms  reach  the  owl  they  enter  the  red  corpuscles.  They  assimilate 
food  material,  and  appear  not  to  migrate  so  frequently  as  the  indifferent 
forms.  They  lose  their  capacity  of  assuming  the  free  trypanosoma 
form,  and  ultimately  their  capacity  of  migration,  so  that  they  are 
found  lying  surrounded  by  the  remains  of  the  last  cell  they  entered. 
The  male  forms  when  they  reach  the  blood  of  the  host  rapidly  die,  and 
the  microgametocytes  are  always  recruited  from  the  indifferent  forms, 
or  from  parthenogenesis  of  the  female  forms. 

It  may  be  said  that  according  to  Schaudinn  the  trypanosomes  gain 
access  to  the  tissues  of  the  mosquito  by  perforating  the  intestinal  wall, 
and,  passing  through  the  body  till  they  reach  the  wall  of  the  pharynx, 
they  burst  through  this,  and  are  in  a  position  to  be  ejected  when  next 
the  insect  bites. 

Such  are  the  views  put  forward  by  this  observer  on  the  cycle 
of  life-history  of  the  Tr.  noctuse.  It  will  be  recognised  that 
the  essential  point  is  the  occurrence,  both  in  the  vertebrate  and 
invertebrate  host,  of  halteridium  stages  alternating  with  those 
in  which  the  trypanosome  form  is  assumed.  It  is  evident  that 
if  this  were  substantiated  important  effects  would  follow  in  our 
views  as  to  the  morphology  not  only  of  the  trypanosome  group, 
but  also  of  that  to  which  the  malarial  parasites  belong. 

Certain  criticisms  of  these  results  have  been  made,  especially  by 
Novy  and  MacNeal,  who  sought  confirmatory  evidence  by  means  of  their 
culture  method.  These  observers  are  of  opinion  that  the  appearances 
described  by  Schaudinn  were  due  to  his  dealing  with  a  mixed  infection 
of  the  owls  by  trypanosomes  on  the  one  hand  and  hsemamcebse  of  the 
halteridium  type  on  the  other.  They  examined  a  very  large  number  of 
different  species  of  birds,  and  established  the  fact  that  infection  with 
halteridium  parasites  on  the  one  hand  and  with  trypanosomes  on  the 
other  is  extremely  common,  and  further,  that  in  the  blood  of  the  same 
bird  both  halteridium  and  trypanosome  infection  could  be  observed. 
The  bird  trypanosomes  could  be  readily  cultivated,  and  it  was  observed 
that  no  cultures  were  obtainable  from  birds  in  which  halteridia  were 
alone  found,  and  further,  that  when  a  trypanosome  isolated  from  a  case 
where  both  forms  of  parasite  had  been  seen  was  injected  into  a  fresh 


550  .  TRYPANOSOMIASIS 

bird,  only  trypanosome  forms  were  found  to  develop  in  its  body.  These 
results  are,  of  course,  not  quite  conclusive,  as  the  halteridium  stage 
might  only  follow  on  the  sexual  portion  of  the  cycle.  In  this  connection, 
however,  the  fact  may  be  mentioned  that  both  with  Tr.  Brucei  and  Tr. 
ugandense  attempts  to  produce  disease  by  means  of  the  contents  of  the 
stomachs  of  infected  insects  have  failed.  Bruce  was  of  opinion  that 
infection  took, place  in  nagana  by  means  of  the  insect  carrying  the 
trypanosomes  in  the  tube  of  its  proboscis,  where  he  observed  them  to  be 
freely  motile  for  forty-eight  hours  after  the  insect  had  fed,  and  with 
regard  to  the  sleeping  sickness  organism  Minchin  held  a  similar  opinion, 
and  showed  that  if  a  glossina  bit  an  infected  animal,  and  then  in  succes- 
sion two  healthy  animals,  only  the  first  of  the  latter  would  contract  the 
disease — the  proljoscis  being  apparently  cleaned  by  the  biting  process. 

Reference  may  here  be  made  to  the  views  put  forward  by 
Schaudinn  regarding  the  relationship  of  certain  spirilla  to  the 
trypanosomes.  In  the  athene  noctiia,  besides  the  Tr.  noctuae 
already  referred  to,  there  is  a  protozoal  parasite  infesting  the 
leucocytes  known  as  spirillum  Ziemanni  or  leucocytozoon 
Ziemanni,  whose  invertebrate  host  is  also  the  culex  pipiens. 
Ziemann  had  described  the  male  and  female  forms  in  the  owl, 
and  microgametes  had  been  observed  forming  from  the  micro- 
gametocytes.  Schaudinn  observed  the  formation  of  an  ookinete 
in  the  mosquito ;  in  certain  cases  this  ookinete  elongates,  and 
the  vermicule  rolls  itself  up  into  a  ball  with  great  proliferation 
of  the  nucleus.  Each  little  nucleus  attaches  to  itself  a  portion 
of  the  protoplasm,  and  becoming  a  miniature  trypanosome, 
swarms  off  and  becomes  free.  These  minute  trypanosomes 
elongate  and  develop  into  typical  spirilla  by  rolling  their  ribbon- 
shaped  bodies  spirally  along  their  longitudinal  axes,  the 
individuals  possessing  male,  female,  or  indifferent  characters, 
just  as  in  Tr.  noctuae.  These  spirilla  multiply  by  longitudinal 
division,  and  often  after  fission  the  two  individuals  remain 
attached  to  each  other  by  their  posterior  ends,  and  in  this  way 
there  is  made  possible  what  is  often  seen  in  spirilla,  namely,  a 
capacity  to  move  in  either  direction.  The  spirilla  often  divide 
so  frequently  that  ultimately  the  individuals  become  invisible  by 
means  of  the  microscope  and  can  only  be  seen  when  lying  in 
clumps.  In  this  stage  Schaudinn  thinks  the  organism  would  be 
able  to  pass  through  a  Chamberland  filter,  and  this  may  be  a 
very  important  observation,  as  throwing  light  on  the  etiology  of 
certain  diseases,  such  as  yellow  fever,  in  which  no  visible  parasites 
have  been  found. 

Schaudinn's  views  on  the  trypanosomal  characteristics  of  Sp.  Ziemanni 
have  raised  important  questions  regarding  the  morphology  of  other 
similar  forms  which  have  been  long  familiar,  such  as  Sp.  Obermeieri, 
and  also  of  the  Spirochsete  pallida  which  Schaudinn  himself  discovered. 


TRYPANOSOMA   LEWISI  551 

It  is  as  yet  too  soon  to  express  any  opinion  on  the  ultimate  effect  of 
these  views.  According  to  Schaudinn  a  trypanosomal  spirillum  consists 
of  a  central  thread  which  represents  the  posterior  nucleus  of  the 
trypanosome  ;  round  this  thread  the  undulating  membrane  is  spirally 
wound,  and  the  principal  nucleus  is  represented  by  minute  chromatin 
dots  sometimes  seen  in  the  course  of  the  spiral.  Whether  all  spirilla 
have  this  structure  must  be  left  for  future  investigation  to  determine. 
Difficulties  arise  with  regard  to  the  significance  of  an  undulatory 
membrane  as  a  spirillary  characteristic  and  also  with  regard  to  the 
terminal  flagellum  which  Schaudinn  himself  found  in  Spirochsete  pallida, 
and  which  he  had  previously  thought  did  not  occur  in  protozoal  spirilla. 
It  is  possible  that  two  groups  of  organisms  have  hitherto  been  classed 
together  under  the  name  spirillum,  and  that  one  of  these  may  still 
have  to  be  placed  with  the  bacteria. 

Trypanosoma  Lewisi. — In  1878  Lewis  described  in  rats  in 
India  the  occurrence  of  the  parasite  which  now  goes  by  his 
name,  and  since  that  time  this  trypanosome  has  been  found  to 
be  very  common  in  the  blood  of  rats  all  over  the  world,  though 
the  percentage  of  animals  affected  varies  in  different  localities. 
The  condition  is  of  great  interest,  as,  though  the  infection  runs 
a  very  definite  course,  it  is  very  rarely  fatal ;  in  fact,  many 
observers  have  been  unable  to  produce  death  by  infecting  even 
large  series  of  animals.  There  is,  however,  little  doubt  that  a 
fatal  issue  does  occur  sometimes  in  young  individuals,  especially 
when  these  are  infected  with  strains  of  the  organism  imported 
from  other  localities.  The  trypanosome,  which  is  actively 
motile,  is  of  the  usual  length  but  is  somewhat  narrow,  and  its 
protoplasm  does  not  contain  any  granules.  It  multiplies  by 
fission,  of  which  Laveran  describes  two  varieties.  In  one,  the 
organism  splits  longitudinally  and  gives  rise  to  smaller  individuals 
than  the  parent.  In  the  other,  the  trypanosome  loses  its 
ordinary  shape  and  becomes  more  oval;  nuclear  division, 
which  is  often  multiple,  then  takes  place,  and  on  subsequent 
division  of  the  protoplasm  a  number  of  small  flagellate  organisms 
result ;  these  last  may  attain  the  full  form  and  size  before 
dividing  again,  or  they  may  divide  when  still  small.  When  a 
rat  is  infected  by  injection  into  the  peritoneum  active  multi- 
plication goes  on  in  the  cavity  for  a  few  days  and  then  comes  to 
an  end.  Very  soon  after  infection  the  organisms  begin  to  appear 
in  the  blood  and  there  rapid  multiplication  occurs,  the  extent 
of  which  is  sometimes  so  great  that  the  trypanosomes  may  seem 
to  equal  the  red  blood  corpuscles  in  number.  The  animal 
usually  shows  no  symptoms  of  illness.  The  infection  goes  on 
for  about  two  months,  and  then  the  organisms  gradually  dis- 
appear from  the  blood.  In  the  great  majority  of  cases  the  rat 
is  now  immune  against  fresh  infection.  If  trypanosomes  be 


552  TRYPANOSOMIASIS 

introduced  into  its  peritoneum  they  are,  according  to  Laveran, 
taken  up  by  mononucleate  phagocytes  and  destroyed.  The 
serum  of  a  rat  which  has  been  infected  shows  agglutinating 
capacities  towards  the  trypanosomes,  causing  them  to  agglomer- 
ate in  rosettes  in  which  the  flagella  are  directed  outwards,  and 
the  serum  of  immune  rats  has  a  certain  degree  of  protective 
action  if  injected  along  with  the  organism  into  a  susceptible 
animal.  As  has  already  been  noted,  this  trypanosome  has  been 
cultivated  on  artificial  media,  on  which  it  multiplies  freely, 
large  numbers  of  small  forms  being  often  produced.  These 
when  injected  into  rats  give  rise  to  the  usual  infection,  but  not 
so  rapidly  as  when  blood  from  an  infected  animal  is  used. 
The  organism  multiplies  at  the  body  temperature,  but  a  lower 
temperature  is  preferable,  and  at  20°  C.  Novy  and  MacNeal 
succeeded  in  carrying  a  growth  through  many  sub-cultures.  This 
trypanosome  is  very  resistant  to  cooling,  and  has  been  exposed 
for  fifteen  minutes  to  the  temperature  of  liquid  air  ( -  191°  C.) 
without  being  killed.  The  means  by  which  the  rat  becomes 
infected  naturally  is  not  known,  but  probably  this  comes  about 
by  the  bite  of  a  flea  or  louse. 

Nagana  or  Tse-tse  Fly  Disease. — This  is  a  disease  affecting 
under  natural  conditions  chiefly  horses,  cattle,  and  dogs  ;  it  is 
prevalent  especially  in  certain  regions  of  South  Africa,  though 
it  probably  may  occur  elsewhere.  In  the  horse  the  chief 
symptoms  are  the  following : — The  animal  is  observed  to  be 
out  of  condition,  its  coat  stares,  it  has  a  watery  discharge 
from  the  eyes  and  nose,  and  the  temperature  is  elevated ; 
swellings  appear  on  the  under  surface  of  the  abdomen  and  in 
the  legs,  it  gradually  becomes  extremely  emaciated  and 
anaemic,  and  dies  after  an  illness  of  from  two  or  three  weeks  to 
two  or  three  months.  In  other  animals  the  symptoms  are  of 
the  same  order,  though  the  duration  of  the  disease  varies  much ; 
thus  in  the  dog  the  illness  does  not  last  more  than  one  or  two 
weeks,  while  in  cattle  it  may  continue  for  six  months.  It  is 
doubtful  whether  a  domestic  animal  attacked  by  the  disease 
ever  recovers.  The  popular  idea  regarding  the  etiology  of  the 
disease  was  that  it  was  contracted  by  animals  passing  through 
certain  rather  restricted  and  sharply  defined  areas  or  belts 
characterised  by  heat  and  damp,  usually  lying  beside  rivers, 
and  always  infested  by  the  tse-tse  fly  (glossina  morsitans),  to  the 
bite  of  which  the  disease  was  attributed ;  in  this  connection  it 
is  important  to  note  that  though  man  is  frequently  bitten  by 
the  tse-tse  fly  he  does  not  contract  nagana.  Modern  know- 
ledge on  the  subject  dates  from  the  discovery  made  by  Bruce 


NAGANA   OR   TSE-TSE   FLY   DISEASE          553 

in  1894  that  the  blood  of  animals  suffering  from  nagaiia 
swarmed  with  a  trypanosome  now  known  as  the  Tr.  Brucei, 
and  in  1895  he  was  instructed  by  the  Governor  of  Natal  to 
undertake  the  investigation  which  led  him  to  work  out  the  true 
etiology  of  the  disease.  It  may  be  said  that  this  research 
forms  the  starting-point  of  the  important  work  done  during 
the  last  decade  with  regard  to  infections  by  trypanosomes. 
In  his  earlier  work  Bruce  found  that  the  parasite  was  present 
in  the  blood  of  every  animal  suffering  from  nagana  and  absent 
from  the  blood  of  healthy  animals  in  the  affected  districts ; 
further,  that  the  fever  which  marks  the  onset  of  the  disease  was 
accompanied  by  the  appearance  of  the  trypanosome  in  the 
blood ;  and  finally,  that  the  transference  of  the  smallest 
quantity  of  blood  from  an  affected  to  a  healthy  animal  origin- 
ated the  disease.  He  then  proceeded  to  investigate  the  part 
played  by  the  tse-tse  fly  in  the  condition.  He  found  that  if 
flies  taken  from  the-  fly  belt  were  transported  to  a  place  where 
nagana  did  not  occur,  kept  for  a  few  days,  and  then  allowed  to 
bite  susceptible  animals,  the  latter  did  not  contract  the  disease — 
this  result  showing  that  it  was  not,  as  had  been  supposed  by 
some,  a  poison  natural  to  the  insect  which  was  the  pathogenic 
agent.  But  if  such  a  fly  was  allowed  to  bite  a  dog  suffering 
from  the  disease  and  then  to  bite  a  healthy  dog,  the  latter 
contracted  the  malady  and  abundant  trypanosomes  were  found 
in  its  blood.  Again,  threads  dipped  in  the  blood  of  an  infected 
animal  and  allowed  to  dry  caused  the  disease  in  healthy  animals 
up  to  but  rarely  beyond  24  hours  after  being  dried  ;  if,  however, 
the  blood  were  kept  moist,  then  it  retained  its  infectiveness  up 
to  between  4  and  7  days;  up  to  46  hours  living  trypano- 
somes could  be  seen  in  the  tube  of  the  fly's  proboscis. 
This  corresponds  roughly  with  what  was  found  regarding  the 
limits  of  the  infectiveness  of  a  fly,  in  that  24  hours  after  it  has 
been  fed  on  an. infected  animal  its  bite  is  usually  innocuous. 
Further,  Bruce  showed  that  infection  did  not  occur  by  any  food 
or  water  partaken  of  by  an  animal  while  going  through  a  fly 
belt,  for  he  took  horses  through  such  a  region  without  allowing 
them  to  eat  or  drink,  and  found  that  they  still  contracted  the 
infection,  if  during  their  few  hours'  journey  through  the  belt 
they  had  been  bitten  by  the  tse-tse  fly.  Finally,  he  showed 
that  if  flies  were  taken  from  an  infected  area  to  a  healthy  one 
a  few  miles  off  and  allowed  at  once  to  bite  infected  animals,  the 
latter  contracted  nagana. 

By  those  experiments  it  was  thus  determined  that  nagana 
could  be  transmitted  by  the  blood  of  the  infected  animal,  that 


554 


TRYPANOSOMIASLS 


is,  without  the  agency  of  the  fly ;  that  the  latter  had  no  inherent 
power  to  produce  the  disease ;  that  it  could,  however,  by 
successively  biting  infected  and  healthy  animals  transmit  the 
disease  to  the  latter ;  and  that  specimens  of  the  insect  caught  in 
infected  areas  harboured  the  parasite  and  were  thus  infective. 
The  question  remained  as  to  how  the  flies  might  become  infected 
in  nature.  It  had  been  observed  that  in  districts  where  the 


FIG.  168. — Trypanosoma  Brucei  from  blood  of  infected  rat.  Note  in  two 
of  the  organisms  commencing  division  of  micronucleus  and  undulating 
membrane.  x  1000. 

tse-tse  fly  lived  the  prevalence  of  the  disease  in  imported  animals 
was  related  to  the  presence  in  the  locality  of  wild  herbivora. 
Bruce  now  found  that,  if  considerable  amounts  of  the  blood 
of  the  latter  were  taken  to  another  locality  and  injected  into 
dogs,  these  in  a  proportion  of  cases  contracted  nagana,  and  from 
this  he  deduced  that  the  wild  animals  harboured  the  parasites 
in  small  numbers  in  their  blood  and  thus  kept  up  the 
possibility  of  infection.  A  further  and  as  yet  unexplained 
fact  was  that  other  blood -sucking  flies  besides  the  tse-tse 


NAGANA   OR   TSE-TSE   FLY   DISEASE          555 

appeared  incapable  of  acting  as  carriers  of  infection.  Bruce's 
work  as  a  whole  pointed  to  the  trypanosome  as  the  cause  of 
nagana,  and  this  has  since  been  finally  established  by  the 
origination  of  the  disease  by  artificial  cultures  of  the  organism. 

The  Tr.  Brucei  (Fig.  168),  according  to  Laveran,  measures 
in  the  horse  from  28-33  //,  long  and  from  1  '5  to  2*5  //,  broad ;  in 
the  rat  and  dog  it  is  somewhat  shorter.  It  is  motile,  but  its 
activity  is  less  than  that  of  Tr.  Lewisi.  When  stained  it 
presents  the  usual  appearances ;  its  posterior  end  is  usually 
blunt,  and  the  body  often  contains  granules  in  the  anterior 
portion  of  its  protoplasm.  It  divides  longitudinally,  and 
according  to  Bradford  and  Plimmer  a  form  of  longitudinal 
conjugation  occurs  in  the  blood.  According  to  the  same 
observers,  it  can  be  kept  alive  for  5-6  days  in  blood  outside 
the  body.  It  is  less  resistant  to  the  action  of  cold  than  Tr. 
Lewisi,  perishing  in  a  few  days  at  5-7°  C.,  but  like  the  other 
organism  it  can  withstand  short  exposures  to  temperatures  down 
to  -191°  C. ;  it  is  quickly  killed  at  44-45°  C.  Novy  and 
MacNeal  succeeded  in  cultivating  this  trypanosome  also, 
though  here  it  was  very  difficult  to  obtain  a  first  growth  from 
the  blood  on  their  blood-agar  medium ;  once  started,  however, 
it  was  kept  alive  through  many  sub-cultures,  the  optimum 
temperature  of  growth  being  25°  C.,  and  it  was  from  these 
sub-cultures  that  the  infection  was  obtained  which  definitely 
proved  the  organism  to  be  the  cause  of  the  disease.  In  cultures, 
as  with  Tr.  Lewisi,  short  forms  occur,  and  there  is  sometimes  a 
rosette  formation  with  the  flagella  directed  outwards  ;  agglutina- 
tion phenomena  are  also  observable  in  defibrinated  blood. 
Under  unfavourable  conditions  involution  forms  occur,  the 
organism  dividing  frequently  to  form  round  flagellated 
individuals. 

Nearly  all  laboratory  animals  are  susceptible  to  infection, 
and  the  duration  of  the  illness  corresponds  to  what  has  been 
observed  in  the  natural  infection  of  these  animals.  The  rat 
has  been  largely  used  for  experiment  and  usually  succumbs  in 
about  ten  days,  there  being  very  few  symptoms  up  till  a  few 
hours  before  death.  A  very  important  fact  has  been  observed 
with  regard  to  this  animal,  namely,  that  individuals  which  have 
gone  through  infection  with  Tr.  Lewisi  and  which  are  immune 
are  still  susceptible  to  the  Tr.  Brucei;  from  this  it  has  been 
deduced  that  the  two  organisms  are  to  be  looked  on  as  distinct 
species. 

Trypanosoma  of  Sleeping  Sickness. — Since  the  year  1800 
the  disease  called  sleeping  sickness,  sleeping  dropsy,  or  negro 


556  TRYPANOSOMIASIS 

lethargy  has  been  recognised  as  prevailing  on  the  West  Coast  of 
Africa  from  the  Senegal  to  Lagos  and  in  the  parts  lying  behind 
the  coast  between  these  regions.  It  has  also  been  found  to  be 
rife  from  Cameroon  to  Angola  and  in  the  Congo  valley,  and  to  a 
less  extent  up  the  Niger  and  its  tributaries.  In  1901  it  began 
to  appear  in  the  Uganda  Protectorate,  and  it  is  in  that  region 
that  the  investigations  have  been  carried  on  which  have  led 
to  a  knowledge  of  its  cause ;  here  it  has  wrought  very  serious 
havoc  amongst  the  native  population.  It  is  characterised 
in  the  early  stages  by  a  change  in  disposition  leading  to 
moroseness,  apathy,  disinclination  for  work  or  exertion,  and 
slowness  of  speech  and  gait.  There  may  be  headache,  indefinite 
pains  about  the  body,  the  evening  temperature  may  be  elevated 
several  degrees,  the  pulse  tends  to  be  soft  and  rapid,  and  in  a 
very  large  number  of  cases  the  superficial  glands  of  the  body  are 
enlarged.  In  a  rapid  case  the  lethargy  becomes  more  pronounced  ; 
fine  tremors,  especially  of  the  tongue  and  arms,  develop ;  pro- 
gressive emaciation  occurs  •  blood  changes  appear,  consisting  of  a 
progressive  diminution  of  the  red  cells  and  of  the  haemoglobin, 
and  of  a  lymphocytosis  in  which  the  percentage  of  both  the 
large  and  small  mononuclear  cells  is  increased,  so  that  the  former 
may  constitute  from  20  to  30  and  the  latter  from  30  to  40  per 
cent  of  all  the  white  cells  present.  As  the  disease  progresses 
the  drowsiness  increases  till  it  deepens  into  a  coma  from  which 
the  individual  cannot  be  roused.  Often  during  the  disease  there 
occur  irregular  cedematous  patches  on  the  skin  and  sometimes 
erythematous  eruptions,  and  effusions  into  the  serous  cavities. 
Not  every  case  runs  a  progressively  advancing  course.  Some- 
times along  with  enlargement  of  glands  the  chief  early  feature 
is  the  occurrence  from  time  to  time  of  attacks  of  fever  which 
may  be  mistaken  for  malaria,  and  from  these  apparently  coin- 
plete  recovery  may  take  place;  recurrence,  however,  follows 
as  a  rule,  and  ultimately  the  typical  terminal  phenomena 
may  commence.  Such  cases  may  go  on  for  years,  and  it  is 
probable  that  many  patients  die  of  pneumonia  without  exhibiting 
typical  manifestations  of  the  malady  from  which  they  really 
suffer.  The  disease  is  an  extremely  fatal  condition,  and  probably 
no  case  where  the  actual  lethargy  is  developed  ever  recovers. 

On  considering  the  disease  from  the  standpoint  of  patho- 
logical anatomy  there  is  little  to  be  said.  As  Mott  described, 
the  most  striking  feature  is  the  presence  of  a  chronic  meningo- 
encephalitis  and  meningo-myelitis.  The  pia-arachnoid  is  some- 
times opaque  and  slightly  thickened  and  may  be  adherent 
to  the  brain,  and  its  vessels  usually  show  some  congestion. 


TRYPANOSOMA   OF   SLEEPING   SICKNESS     557 

The  sub-arachnoid  fluid  is  sometimes  in  excess  and  occasionally 
may  even  be  purulent.  The  membranes  of  the  spinal  cord 
show  similar  changes.  The  chief  other  feature  is  the  presence 
of  enlarged  lymphatic  glands  in  the  body,  but  otherwise  there  is 
nothing  special  to  note.  With  regard  to  the  microscopic 
changes  the  chief  feature,  according  to  Mott,  is  a  proliferation 
and  overgrowth  of  the  neuroglia  cells,  especially  of  those  which 


Fm.  169. — Trypanosoma  gambiense  from  blood  of  guinea-pig.      x  1000. 

are  related  to  the  sub-arachnoid  space  and  the  perivascular  lymph 
spaces,  with  accumulation  and  probably  proliferation  of  lympho- 
cytes in  the  meshwork.  He  further  points  out  that  the  changes 
in  the  lymph  glands  are  of  similar  nature  and  resemble  the  in- 
filtration of  the  perivascular  lymphatics  of  the  central  nervous 
system.  These  changes  are  specially  significant  in  view  of  the 
lymph ocytosis  present  in  the  blood,  which  has  already  been 
noted,  and  which  so  often  occurs  in  protozoal  infections.  In  the 
nervous  structures  there  is  comparatively  little  change,  there 
being  merely,  according  to  Mott,  some  atrophy  of  the  dendrons 


558  TRYPANOSOMIASIS 

of  the  nerve  cells,  a  diminution  of  Nissl's  granules,  and  an 
excentricity  of  the  nucleus. 

Trypanosoma  gambiense. — Before  going  further  we  must 
refer  to  the  observation  of  a  trypanosome  in  the  blood  of  persons 
not  evidently  suffering  from  sleeping  sickness.  The  first  case  of 
this  was  recorded  by  Dutton  in  1901,  the  patient  being  a 
European  then  living  at  Bathurst  on  the  Gambia.  The  progress 
of  the  disease  was  here  very  slow,  and  was  characterised  by 
general  wasting  and  weakness,  irregular  rises  of  temperature, 
local  oedemas,  congested  areas  of  the  skin,  enlargement  of  spleen, 
and  increased  frequency  of  pulse  and  respiration ;  death  occurred 
a  year  after  the  case  came  under  observation  after  an  access  of 
fever,  and  a  striking  fact  was  the  absence  of  any  gross  causal 
lesion.  During  the  time  the  patient  was  under  observation 
trypanosomes  were  repeatedly  demonstrated  in  the  peripheral 
blood,  and  they  also  developed  in  the  bodies  of  monkeys 
and  white  rats  inoculated  with  the  blood.  Pursuing  further  in- 
quiries, Dutton  and  Todd  demonstrated  similar  parasites  in  other 
Europeans  and  in  several  natives  in  the  Gambia  region,  whilst 
about  the  same  time  Manson  reported  a  case  of  the  same  kind  in 
the  wife  of  a  missionary  on  the  Congo.  It  thus  came  to  be 
recognised  that  in  man  there  occurred  a  disease  having  characters 
somewhat  resembling  nagana  and  in  which  trypanosomes  could 
be  demonstrated  in  the  blood,  and  this  was  usually  referred  to  as 
human  trypanosomiasis,  or  trypanosoma  fever, — the  trypanosome 
being  named  the  Tr.  gambiense. 

Relation  of  Trypanosomes  to  Sleeping  Sickness.  —  Several 
views  as  to  the  etiology  of  this  disease  had  been  advanced.  A 
Portuguese  Commission  in  1902  described  a  diplococcus  tending 
to  grow  in  chains  which  they  isolated  from  the  cerebro-spinal 
fluid  taken  from  cases  during  life,  and  to  which  they  were 
inclined  from  the  constancy  of  its  occurrence  to  attribute  a 
causal  role.  The  seriousness  of  the  epidemic  in  Uganda  had  led 
the  Royal  Society  of  London  in  1902,  at  the  instigation  of  the 
Foreign  Office,  to  despatch  a  Commission  to  investigate  the 
condition  on  the  spot.  Soon  after  commencing  work,  Dr. 
Castellani  found  in  some  cases  in  the  cerebro-spinal  fluid, 
especially  when  this  was  centrifugalised,  living  trypanosomes 
resembling  the  Tr.  gambiense ;  he  also  found  in  80  per  cent  of 
the  cases  post  mortem  a  coccus  resembling  if  not  identical  with 
that  observed  by  the  Portuguese  Commissioners.  At  first 
Castellani  was  inclined  to  look  on  the  presence  of  the  protozoan 
as  accidental,  but  Colonel  Bruce  on  going  out  with  Nabarro 
and  Greig  in  1903  to  pursue  the  work  of  the  Commission 


TRYPANOSOMA   OF   SLEEPING   SICKNESS      559 

realised  the  significance  of  the  observation,  urged  Castellani  to 
further  inquiries,  which  he  himself  continued  after  the  departure 
of  the  latter,  with  the  result  that  in  a  series  of  examinations 
carried  out  in  several  infected  localities,  the  trypanosome  was 
demonstrated  in  every  case  of  the  disease.  This  work  formed 
the  starting-point  for  inquiries,  the  results  of  which  make  it 
practically  certain  that  the  parasite  is  the  causal  agent  of  the 
condition.  The  organisms  were  not  demonstrable  in  the  cerebro- 
spinal  fluid  of  patients  dying  of  other  diseases  in  the  sleeping  sick- 
ness area.  On  the  other  hand,  it  was  found  that  if  cerebro-spinal 
fluid  withdrawn  from  cases  of  the  disease  was  injected  into  monkeys 
(especially  macacus  rhesus)  trypanosomes  appeared  in  the  blood, 
and  in  many  cases  in  three  or  four  months  the  animals  died  of  an 
illness  indistinguishable  from  sleeping  sickness  and  with  the  p^ra- 
sites  in  the  central  nervous  system.  It  was  further  found  that  in 
the  parts  round  the  north  end  of  Victoria  Nyanza  where  sleeping 
sickness  was  rife,  the  distribution  of  the  disease  exactly  corre- 
sponded with  the  distribution  of  a  blood-sucking  insect,  the 
glossina  palpalis,  a  species  closely  allied  to  the  glossina  morsitans 
of  nagana.  It  was  found  that,  when  one  of  these  flies  was  fed  on 
a  sleeping  sickness  patient  and  then  allowed  to  bite  a  monkey, 
frequently  trypanosomes  appeared  in  the  animal's  blood,  and 
that  when  fresh  flies  caught  in  the  sleeping  sickness  area  were 
placed  on  a  monkey  a  similar  occurrence  took  place. 

The  trypanosome  of  sleeping  sickness  is  17-28  /x,  long  and 
l'4-2  /JL  broad  (Fig.  169)  (when  about  to  divide  it  is  both  longer 
and  broader).  According  to  Laveran  the  free  part  of  the 
flagellum  often  equals  a  fourth  of  the  whole  length,  but  occasion- 
ally the  body  protoplasm  extends  quite  to  the  end  of  the 
organism.  The  undulating  membrane  is  narrow,  and  the 
posterior  end  may  be  either  sharp  or  blunt.  The  trypanosome 
contains  the  macro-  and  micronucleus  characteristic  of  the  group, 
and  the  protoplasm  often  shows  chromatin  granules.  Castellani 
attached  great  importance  to  a  vacuole  often  seen  in  the  neigh- 
bourhood of  the  micronucleus,  but,  as  stated  above,  Laveran 
holds  this  to  be  an  artefact.  The  organism  divides  longitudin- 
ally in  the  usual  manner,  and  often  two  can  be  seen  to  approach 
each  other  and  lie  more  or  less  side  by  side,  but  whether  this 
indicates  conjugation  or  not  is  not  known.  The  organism  does 
not  usually  long  survive  removal  from  the  body,  but  it  has  been 
found  to  be  motile  for  nineteen  days  when  kept  on  rabbit-blood 
agar  at  22°  C.  As  we  have  said,  when  Tr.  ugandense  is  in- 
oculated into  monkeys  they  often  contract  an  illness  which 
ultimately  presents  the  features  of  typical  sleeping  sickness. 


560  TRYPANOSOMIASIS 

Inoculation  of  other  species  of  animals  is  not  usually  so 
successful,  though  in  nearly  every  case,  e.g.  in  the  gui'nea-pig,  a 
proliferation  of  the  parasite,  as  indicated  by  its  appearing  in  the 
blood,  takes  place  ;  but  often  either  no  disease  occurs  or  this  runs 
a  very  chronic  course.  The  relative  insusceptibility  of  animals, 
especially  of  the  dog,  to  the  Tr.  ugandense  is  taken  as  evidence 
that  this  organism  is  essentially  different  from  Tr.  Brucei. 

By  means  of  microscopic  examination  the  organisms  may  be 
found  in  the  cerebro-spinal  fluid,  the  blood,  or  the  juice  of 
glands.  In  the  case  of  the  first  about  10  c.c.  of  the  fluid  is  to 
be  centrifuged  for  fifteen  minutes  and  the  deposit  placed  under 
a  cover-glass  for  examination ;  it  is  better  to  make  a  little  cell 
on  a  slide  by  painting  a  ring  of  ordinary  embedding  paraffin, 
to  place  the  droplet  of  fluid  in  its  centre,  and  to  support  the  cover- 
glass  on  the  paraffin  ;  in  this  way  injury  to  the  delicate  structure 
of  the  organism  is  avoided.  In  fresh  cerebro-spinal  fluid  the 
trypanosomes  can  be  seen  to  be  actively  motile ;  the  number  in 
which  they  occur  varies  very  much,  and  the  same  is  true  to  a 
greater  degree  of  the  blood,  in  which  they  are,  however,  usually 
very  scanty.  With  regard  to  the  examination  of  the  blood 
Bruce  and  Nabarro  state  that  it  is  difficult  by  ordinary  centri- 
fugalisation  to  concentrate  the  organisms,  as  they  are  not  readily 
precipitated.  They  accordingly  recommend  that  the  blood  be 
mixed  with  citrate  of  sodium  solution  (equal  parts  of  blood  and  of 
a  one  per  cent  citrate  solution)  and  centrif ugalised  for  ten  minutes, 
that  the  plasma  be  removed  and  centrifugalised  afresh  for 
the  same  time,  and  that  this  be  repeated  three  times,  the  deposit 
from  each  centrifugalisation  after  the  first  being  carefully 
examined.  Greig  and  Gray  have  insisted  that  the  examination 
of  the  glands  in  a  suspected  case  forms  the  most  ready  means  of 
arriving  at  a  diagnosis,  and  this  opinion  has  found  strong 
support  from  the  work  of  Button  and  Todd.  The  method  is  to 
push  a  hypodermic  needle  into  the  gland,  suck  up  a  little  of  the 
juice,  and  blow  it  out  on  to  a  slide.  In  all  cases  where  films  of 
any  kind  are  to  be  prepared  the  staining  methods  of  Leishman 
or  Giemsa  are  to  be  recommended.  Often  in  cerebro-spinal  fluid 
and  gland  juice  the  staining  of  the  chromatin  is  difficult,  but 
good  preparations  are  obtained  by  the  procedure  recommended 
by  Leishman  for  studying  the  parasite  in  sections  (p.  545). 

Greig  and  Gray  have  studied  the  trypanosome  in  the  body 
of  the  glossina.  They  found  evidence  of  its  multiplying  in  the 
stomach  of  the  insect,  and  it  also  was  seen  to  undergo  changes 
not  elsewhere  observed.  These  consisted  in  alterations  in  the 
position  of  the  micronucleus,  which  often  became  anterior  to  the 


TRYPANOSOMA   OF   SLEEPING    SICKNESS      561 

macronucleus ;  there  also  occurred  rosettes,  consisting  of  from 
four  to  twenty  individuals  attached  by  their  posterior  extremities. 
Oval  forms  were  also  observed.  It  was  found  that  monkeys 
could  not  be  inoculated  with  the  trypanosomes  from  the  stomach 
of  the  fly,  and  this  observation  corresponds  with  what  Bruce 
found  to  be  true  of  the  trypanosome  of  nagana  in  glossina 
morsitans. 

Early  in  the  Uganda  investigations  the  question  arose  as  to 
whether  the  trypanosoma  of  sleeping  sickness  was  different  from 
Tr.  gambiense.  This  was  forced  on  the  inquirers  by  the  fact 
that  a  very  large  proportion  of  the  natives  in  the  sleeping 
sickness  area  were  found  to  harbour  trypanosomes  in  their 
blood,  although  not  apparently  suffering  from  the  disease. 
Several  cases  were  carefully  examined  in  which  trypanosomes 
were  constantly  present  in  the  blood,  but  in  which  the  patients 
from  time  to  time  suffered  from  fever,  and  during  these  pyrexial 
periods  trypanosomes  were  found  in  the  cerebro- spinal  fluid. 
It  was  suggested  that  these  cases  were  on  the  way  to  develop 
sleeping  sickness.  A  very  important  observation  was  that 
while  in  sleeping  sickness  areas  a  large  proportion  of  the  native 
population  harboured  trypanosomes,  this  was  not  the  case 
where  sleeping  sickness  did  not  occur.  Further,  it  was  found 
that  trypanosomes  from  the  cerebro -spinal  fluid  of  sleeping 
sickness  cases  and  from  the  blood  of  persons  harbouring  try- 
panosomes, but  not  suffering  from  disease  symptoms,  gave  rise 
in  monkeys  to  the  same  group  of  chronic  effects  which  resembled 
the  last  stages  of  the  disease  in  man.  These  facts  led  the 
Commissioners  to  incline  to  the  idea  that  trypanosoma  fever 
and  sleeping  sickness  are  due  to  the  same  cause,  and  represent 
different  stages  of  the  same  disease.  It  has  already  been 
pointed  out  that  a  fatal  termination  can  occur  in  trypanosoma 
fever  by  an  acute  febrile  attack  or  from  intercurrent  disease, 
and  thus  the  terminal  lethargic  stage  may  only  develop  in  a 
certain  proportion  of  cases.  The  view  of  the  identity  of  the 
two  conditions  has  continued  to  gain  ground.  The  best 
authorities  are  agreed  that  morphologically  no  difference 
between  the  two  organisms  can  be  recognised,  and  the  con- 
tinued observation  of  prolonged  cases  of  trypanosoma  fever, 
both  in  Uganda  by  Greig.  and  Gray  and  in  this  country 
by  Manson,  has  shown  that  sometimes  the  termination  of  a 
case  is  by  the  onset  of  typical  sleeping  sickness. 

The  prevalence  of  trypanosomes  in  the  blood  of  apparently 
healthy  natives  has  raised  the  question  of  the  possibility  of 
tolerance  existing  and  of  immunity  being  established.  It  is 
36 


562  TRYPANOSOMTASIS 

possible  that  both  phenomena  occur,  that  not  every  infection 
results  in  multiplication  of  the  parasite  in  the  body  of  the 
victim,  and  that  in  certain  cases  where  multiplication  does 
occur  a  resistance  is  developed  which  enables  the  body  to  kill 
the  parasites.  The  occurrence  of  the  mononuclear  reaction  is 
here  significant ;  it  has  been  suggested  that,  when  this  resist- 
ance is  weak,  the  organism  gains  entrance  to  the  spinal  canal, 
and  that  then  sleeping  sickness  results. 

The  whole  of  the  recent  work  on  the  disease  is  of  the  highest 
interest  and  importance.  Short  of  the  prolonged  subculture  of 
the  parasite  and  the  reproduction  of  the  disease  by  such  cultures, 
the  strongest  evidence  may  be  said  to  exist  that  the  Tr. 
ugandense  is  the  cause  of  sleeping  sickness. 

Other  Pathogenic  Trypanosomata. — It  is  beyond  the  scope 
of  this  work  to  deal  at  length  with  the  other  diseases  of  animals 
caused  by  trypanosomes.  The  chief  of  these  have  been 
mentioned  in  the  opening  paragraph,  but  it  may  be  said  that 
many  others  have  been  described  in  various  species  of  mammals, 
birds,  and  fishes,  and  that  these  are  spread  either  by  flies  or  by 
leeches.  The  most  interesting  of  those  mentioned  is  Dourine, 
a  condition  resembling  in  many  ways  nagana.  It,  however, 
presents  this  peculiarity,  that  infection  does  not  take  place  by 
an  intermediate  host,  but  apparently  directly  through  coitus,  as 
it  occurs  only  in  stallions  and  in  mares  covered  by  these. 

In  several  of  the  trypanosomal  infections  of  animals  it 
appears  as  if,  as  in  the  case  of  Tr.  Lewisi,  the  animal  suffers 
little  inconvenience  from  the  presence  of  the  parasite  in  its 
blood,  and  the  view  has  even  been  put  forward  that  with  all 
pathogenic  trypanosomes  there  exists  a  host  which  carries  the 
organism  without  being  affected  by  its  presence  more,  for 
example,  than  is  the  rat  by  Tr.  Lewisi.  Though  no  opinion 
can  be  expressed  on  this  point,  it  is  necessary  to  bear  the  fact 
in  mind  that  either  natural  or  acquired  immunity  can  exist 
against  such  protozoa.  Not  only  is  this  important  from  the 
point  of  view  of  the  investigation  of  the  conditions  under  which 
such  tolerance  arises,  but  also  from  the  bearing  which  the 
existence  of  this  tolerance  may  have  on  the  spread  in  nature  of 
the  parasites  to  a  susceptible  species  from  immune  animals  which 
still  harbour  trypanosomes  in  their  blood.  We  are,  however, 
as  yet  quite  ignorant  of  many  of  the  processes  at  work  in  the 
body  during  a  trypanosomal  infection,  and  of  the  causes  of  the 
symptoms  and  other  morbid  effects. 


KALA-AZAR  563 

KALA-AzAR. 

(Synonyms  :  Cachectic  Fever,  Dum-Dum  Fever,  Non-malarial 
Remittent  Fever.) 

Leishman  noticed  in  several  soldiers  invalided  from  India  for 
remittent  fever  and  cachexia  that  the  most  careful  examination 
of  the  blood  failed  to  reveal  the  presence  of  the  malarial 
parasite.  From  the  fact  that  such  patients  had  almost  invari- 
ably been  quartered  during  their  service  at  Dum-Dum,  an 
unhealthy  cantonment  near  Calcutta,  he  suspected  he  had 
to  deal  with  an  undescribed  disease.  In  1900  he  noticed  in 
the  spleen  of  such  a  case  peculiar  bodies  which,  from  comparison 
with  certain  appearances  found  in  degenerating  forms  of  Tr. 
Brucei,  he  suggested  might  be  trypanosomes,  and  on  publishing 
his  observations  in  1903  he  put  forward  the  view  that 
trypanosomiasis  might  prevail  in  India  and  account  for  the 
aberrant  cases  of  cachexial  fever  met  with  there.  Soon  after 
Leishman's  paper  appeared,  his  observations  were  confirmed  in 
India  by  Donovan,  and  the  bodies  associated  with  the  disease 
are  usually  called  the  "  Leishman  "  or  the  "  Leishman-Donovan  " 
bodies.  They  were  found  by  Bentley,  and  later  by  Rogers,  in 
the  disease  known  in  Assam  as  Kala-azar,  the  pathology  of 
whi'ch  had  long  puzzled  those  who  had  worked  at  it,  from 
the  fact  that,  while  it  resembled  malaria  in  many  ways,  no 
parasite  could  be  demonstrated  to  occur  in  those  suffering 
from  it. 

Kala-azar  (or  "black  disease," — so  called  from  the  hue 
assumed  by  chocolate-coloured  patients  suffering  from  it)  has 
been  known  since  1869  as  a  serious  epidemic  disease  in  Assam, 
where  it  has  spread  from  village  to  village  up  the  Brahmaputra 
valley.  The  disease  is  now  known  to  occur  in  various  sub- 
tropical centres  south  of  the  forty-ninth  parallel — cases  where 
the  Leishman  bodies  have  been  found  having  been  met  with  in 
many  parts  of  India,  China,  North  Africa,  and  Arabia.  The 
disease  is  characterised  by  fever  of  a  very  irregular  type, 
by  progressive  cachexia,  and  by  anaemia  associated  with 
enlargement  of  the  spleen  and  liver,  and  often  with  ulcers  of 
the  skin  and  dropsical  swellings.  Rogers  has  pointed  out  that 
there  occurs  a  leucopenia  which  differs  from  that  of  malaria  in 
that  it  is  almost  always  more  marked, — the  leucocytes  usually 
numbering  less  than  2000,  and  further  in  that  they  are  always 
reduced  in  greater  ratio  than  the  red  corpuscles,  which  condition, 
again,  does  not  occur  in  malaria.  The  disease  is  chronic, 


564 


KALA-AZAR 


often  going  on  for  several  years,  and  is  extremely  fatal, — in 
fact,  it  is  difficult  to  say  if  recovery  can  ever  take  place. 
Post  mortem,  there  is  little  to  note  beyond  the  enlargement  of 
the  liver  and  spleen,  but  in  the  intestine,  especially  in  the 
colon,  there  are  often  large  or  small  ulcers,  and  there  is  evidence 
of  proliferation  in  the  bone  marrow,  the  red  marrow  encroaching 
on  the  yellow. 

In  a  film  made  from  the  spleen  and  stained  by  Leishman's 


FIG.  170. — Leishman- Donovan  bodies  from  spleen  smear.      x  1000. 

stain,  the  characteristic  bodies  can  be  readily  demonstrated 
(Fig.  170).  They  are  round,  oval,  or,  as  Christophers  has 
pointed  out,  cockle-shell  shaped,  and  usually  2*5  to  3  '5  p  in 
diameter,  though  smaller  forms  occur.  The  protoplasm  stains 
pink,  or  sometimes  slightly  bluish,  and  contains  two  bodies 
taking  on  the  bright  red  colour  of  nuclear  matter  when  stained 
by  the  Romanowsky  combination.  The  larger  stains  less 
intensely  than  the  smaller,  is  round,  oval,  heart-shaped,  or 
bilobed,  and  lies  rather  towards  the  periphery  of  the  body — in 
the  region  of  the  "hinge"  in  the  cockle-shaped  individuals. 


KALA-AZAR  565 

The  other  chromatin  body  is  usually  rod-shaped,  and  is  set 
perpendicularly  or  at  a  tangent  to  the  larger  mass,  with  which 
only  exceptionally  it  appears  to  be  connected.  Usually  the 
protoplasm  contains  one  or  two  vacuoles.  Though  in  spleen 
smears  many  free  bodies  are  seen,  the  study  of  sections  shows 
that  ordinarily  their  position  is  intra-cellular, — the  cells  con- 
taining them  being  of  a  large  mononuclear  type  (Fig.  171).  The 
view  held  is  that  on  their  entering  the  circulation  they  are 
taken  up  by  the  mononuclear  leucocytes  and  by  such  cells  as 
the  endothelial  lining  of  the  splenic  sinuses  or  by  those  lining 
capillaries  or  lymphatics,  that  in  these  cells  multiplication  takes 
place — it  may  be  to  such 
an  extent  as  to  rupture 
the  cell, — and  that  if 
thus  the  bodies  become 
free,  they  are  taken  up 
by  other  cells  and  the 
process  is  repeated.  The 
clusters  of  bodies  some- 
times  seen  in  smears  are 
probably  held  together 
by  the  remains  of  rup- 
tured  phagocytes.  In 
capillaries  the  endothelial 
cells  after  phagocyting 
the  bodies  probably  be- 
come detached  from  the 

capillary   wall,    as   they 

„  J         .    '         -i    £  FIG.  1/1. — Leishman-Douovan  bodies  within 

are   otten   observed  tree  endothelial  cell  in  spleen,      x  1000. 

in    the    lumen    of    the 

vessel,  —  this  being  well  seen  in  the  hepatic  capillaries. 
In  the  body  generally  the  parasites  are  found  in  greatest 
abundance  in  the  spleen,  liver,  and  bone  marrow,  and  also  in 
mesenteric  glands,  especially  in  those  draining  one  of  the 
intestinal  ulcers ;  less  frequently  they  occur  in  the  skin  ulcers, 
and  in  other  parts  of  the  body.  Whether  they  can  be  demon- 
strated microscopically  in  the  blood  is  disputed.  Donovan 
described  them  as  occurring  in  the  blood,  and  also  as  being 
present  within  red  blood  corpuscles,  but  though  Laveran  agreed 
with  Donovan's  description,  the  observation  has  not  been 
confirmed  by  other  observers. 

In  the  body  the  parasite  multiplies  by  simple  fission,  both 
nuclei  dividing  amitotically,  and  two  new  individuals  being 
formed ;  but  sometimes  a  multiple  division  takes  place,  each 


566  KlLA-AZAR 

nucleus  dividing  several  times  within  the  protoplasm  and  a 
corresponding  number  of  new  parasites  resulting. 

In  view  of  Leishman's  original  opinion  an  extremely  important 
discovery  was  made  by  Rogers  and  later  confirmed  by  Leishinan 
himself,  to  the  effect  that  in  cultures  a  flagellate  organism 
developed  from  the  Leishman-Donovan  body.  Cultivation  was 
effected  by  taking  spleen  juice  containing  the  parasite,  placing  it 
in  10  per  cent  sodium  citrate  solution  and  keeping  at  17-24°  C. 
Under  such  conditions  there  occurs  an  enlargement  of  the 
organism,  but  especially  of  the  larger  nucleus.  This  is  followed 
by  the  appearance  of  a  pink-staining  vacuole  in  the  neighbourhood 
of  the  smaller  nucleus.  Along  with  these  changes,  in  from  24  to 
48  hours  the  parasite  becomes  elongated  and  the  smaller  nucleus 
and  its  vacuole  move  to  one  end ;  from  the  vacuole  there  then 
appears  to  develop  a  red-staining  flagellum,  which  when  fully 
formed  seems  to  take  its  origin  from  the  neighbourhood  of  the 
small  nucleus.  The  body  of  the  parasite  is  now  from  20-22  //, 
long  and  3-4  /*  broad,  with  the  flagellum  about  22  \L  long.  The 
whole  development  occupies  about  96  hours.  The  formation  of 
an  undulating  membrane  was  not  observed,  and,  although  the 
flagellated  organism  moved  flagellum  first,  like  a  trypanosome, 
it  is  evident  that  here  the  relationship  of  the  micronucleus  is 
different  as  this  structure  lies  anterior  to  the  macronucleus. 
In  his  cultures,  which  kept  alive  for  four  weeks,  Leishinan  made 
a  still  further  important  observation.  In  certain  of  the  flagellate 
forms  he  saw  chromatin  granules  develop  in  the  protoplasm  often 
in  couples,  a  larger  and  a  smaller.  There  then  occurred  a  very 
unequal  longitudinal  division  of  the  protoplasm,  and  a  hair-like 
undulating  individual  containing  one  of  the  pairs  of  chromatin 
granules  would  be  split  off.  At  first  these  would  be  non- 
flagellate,  but  later  a  red-staining  flagellum  would  appear  at  one 
end.  The  analogies  between  these  observations  and  those  of 
Schaudinn  (v.  p.  550)  on  the  relations  of  spirochsetes  to 
trypanosomes  will  be  at  once  apparent;  the  further  develop- 
ment of  these  spirillary  forms  in  Leishman's  organism  could 
not,  however,  be  traced. 

The  facts  just  detailed  have  caused  considerable  discussion  as 
to  the  classification  of  the  organism,  which  now  usually  goes  by 
the  name  Leishmania  donovani,  originally  given  to  it  by  Ross. 
According  to  one  view,  it  is  to  be  looked  on  as  a  trypanosome, 
and  although,  as  we  have  noted,  its  flagellated  form  differs  from 
the  typical  trypanosoma  form,  it  bears  considerable  resemblance 
to  the  members  of  this  group,  and  as  Leishman  has  pointed  out, 
his  cultures  may  not  represent  the  full  development  of  the 


KALA-AZAR  567 

organism  in  the  trypanosoma  direction.  Others  have  looked 
on  it  as  a  piroplasma,  but  Minchin  suggests  that  in  the  present 
incomplete  state  of  knowledge  it  may  be  well  to  place  it  in  a 
provisional  genus,  Leishmania,  of  the  flagellata.  In  this  genus 
there  would  be  at  present  two  species,  the  Leishmania  donovani, 
and  the  organism  seen  in  Dehli  sore,  Leishmania  tropica, 
presently  to  be  alluded  to. 

The  question  arises,  given  that  the  Leishmania  donovani  is 
the  cause  of  kala-azar,  how  is  infection  spread1?  On  this  we 
have  as  yet  no  certain  information.  The  fact  that  in  some 
centres  of  the  disease  natives  who  are  supplied  with  good  water 
are  less  liable  than  those  who  rely  on  the  ordinary  polluted 
native  cisterns,  has  led  to  the  opinion  that  water  may  be  the 
carrier  of  infection.  On  the  other  hand,  the  possible  relation- 
ship of  the  organism  to  the  trypanosomata  naturally  suggests 
the  idea  of  an  insect  as  an  intermediary,  and  Rogers  has  brought 
forward  some  evidence  that  the  bed-bug  is  the  extra-human  host, 
but  the  organism  has  not  as  yet  been  demonstrated  in  the  body 
of  this  insect.  It  has  been  objected  to  the  theory  of  an  insect 
carrier  that  the  organism  probably  does  not  occur  in  the  blood, 
but  it  has  been  pointed  out  that  invisible  spirillary  forms  may 
be  the  instruments  of  infection,  and  that  such  may  exist  in  the 
blood.  It  may  be  said  here  that  all  attempts  to  communicate 
the  disease  to  animals  have  been  hitherto  unsuccessful. 

With  regard  to  kala-azar  as  a  whole,  we  may  say  that  we  are 
dealing  with  a  disease  fairly  widespread  in  various  sub-tropical 
regions,  which  is,  there  is  every  reason  to  believe,  a  separate 
entity.  All  attempts  to  include  it  among  the  malarial  cachexias, 
which  clinically  it  so  much  resembles,  have  failed.  In  this  atypical 
cachexial  fever  there  is  always  present  a  parasite  of  very  special 
characters  belonging  or  closely  allied  to  a  group  which  contains 
many  varieties  capable  of  giving  rise  to  similar  diseases. 
Beyond  this  we  cannot  go,  but  at  present  we  must  admit  that 
there  is  strong  presumptive  evidence  of  the  parasite  described 
being  the  cause  of  the  disease. 

Methods  of  Examination. — The  Leishmania  donovani  can 
be  readily  seen  in  films  or  sections  of  the  organs  in  which  we 
have  mentioned  its  occurrence.  These  should  be  stained  by  the 
Romanowsky  stains.  Fluid  taken  from  the  enlarged  spleen  during 
life  may  be  examined,  but  it  is  probable  that  in  this  disease 
puncture  of  the  spleen  may  not  be  a  very  safe  operation,  as 
death  from  haemorrhage  from  this  organ  is  a  not  uncommon 
natural  terminal  event.  During  life  the  main  points  on  which 
a  pathological  diagnosis  may  be  based  are  the  absence  of 


568  KALA-AZAR 

malarial  parasites  from  the  blood,  and  the  features  of  the 
leucopenia  which  have  been  alluded  to. 

Delhi  Sore. — In  various  tropical  and  sub-tropical  regions 
there  is  widely  prevalent  a  variety  of  very  intractable  chronic 
ulceration  which  goes  by  various  names  in  different  parts  of 
the  world,  Delhi  sore,  tropical  ulcer,  Aleppo  boil,  etc.  Various 
views  have  been  held  as  to  the  pathology  of  the  condition,  but 
the  work  of  J.  H.  Wright,  which  has  been  confirmed  by  other 
observers,  makes  it  extremely  probable  that  a  protozoal  parasite 
is  concerned  in  its  etiology.  In  the  discharge  from  the  ulcer 
and  in  sections  of  a  portion  of  tissue  excised  from  a  case  coming 
from  Armenia,  Wright  observed  great  numbers  of  round  or  oval, 
sharply  defined  bodies,  2-4  //.  in  diameter.  When  stained  by  a 
Komanowsky  combination  there  was  found  to  be  a  peripheral 
portion  coloured  a  pale  blue  and  a  central  portion  tending  to  be 
unstained ;  there  were  also  two  chromatin  bodies,  one  larger, 
occupying  a  fourth  or  a  third  of  the  whole  and  situated  in  the 
periphery,  another  smaller,  round  or  rod-shaped,  and  of  a  deeper 
colour  than  the  larger  mass.  It  was  found  that  the  bodies 
were  usually  intra-cellular  in  position  in  the  lesion,  as  many  as 
twenty  being  in  one  cell,  and  that  the  type  of  cell  containing 
them  was,  as  in  kala-azar,  that  derivable  from  endothelial  tissues. 

There  can  be  little  doubt  that  these  bodies  are  of  the  same 
type  as  those  occurring  in  kala-azar,  and  the  question  of  the 
identity  of  the  two  parasites  has  been  raised.  At  present  the 
tendency  is  to  regard  them  as  distinct.  As  we  have  seen, 
although  skin  ulcers  are  common  in  kala-azar,  it  is  difficult  to 
find  the  parasite  in  this  lesion  of  the  disease,  while,  on  the 
other  hand,  in  Wright's  case  at  least  the  number  of  organisms 
present  in  the  ulcer  was  enormous.  Provisionally  Minchin 
calls  this  parasite  the  Leishmania  tropica  and  includes  it  as  the 
second  species  of  the  genus  Leishmania. 

PIROPLASMOSIS. 

Up  to  the  present  no  human  disease  has  been  proved  to  be  associated 
with  the  presence  of  piroplasmata.  The  observations  of  Donovan, 
which  seemed  to  indicate  that  the  parasite  of  kala-azar  might  be  found 
within  the  red  blood  corpuscles,  and  which  led  Laveran  to  denominate 
the  Leishmania  donovani  the  piroplasma  donovani,  have,  as  already 
indicated,  not  been  confirmed  ;  the  same  is  true  of  the  association  of 
piroplasms  with  the  occurrence  of  the  Rocky  Mountain  spotted  fever 
sometimes  prevalent  in  Montana.  But  several  important  diseases  of  the 
lower  animals  are  almost  certainly  caused  by  protozoan  parasites  of  this 
group,  and  a  short  account  of  the  organisms  may  be  given. 

The  piroplasmata  are  pear-shaped  unicellular  organisms  about  1-1 '5  ju, 


PIROPLASMOSIS  569 

long  and  varying  in  breadth.  The  peripheral  part  is  denser  than  the 
central,  which  often  appears  as  if  vacuolated,  and  at  the  broad  end  there 
is  a  well-staining  chromatin  mass.  Sometimes  irregular  and  ring-,  rod-, 
or  oval-shaped  individuals  occur.  The  organisms  are  found  within  the 
red  blood  corpuscles  of  the  infected  animal  and  also  free  in  the  blood. 
In  the  former  situation  there  is  sometimes  only  one  within  a  cell,  but 
the  numbers  vary  under  different  circumstances  and  in  different  species. 
Multiplication  takes  place  by  fission,  and  the  new  individuals,  remaining 
for  longer  or  shorter  times  in  apposition,  account  for  some  of  the 
appearances  seen  in  cells.  Especially  in  the  forms  free  in  the  blood 
pseudopodial  prolongations  of  the  protoplasm,  usually  from  the  pointed 
end,  are  developed,  and  it  may  be  by  means  of  such  pseudopodia  that 
entrance  to  the  red  cells  is  obtained.  Infection  is  usually  carried  from 
infected  animals  by  means  of  ticks.  In  one  case  Koch  has  described  the 
development  in  the  organism,  in  the  stomach  of  the  tick,  of  spiked  proto- 
plasmic processes  sprouting  out  from  the  broad  end  of  the  piroplasm, 
and  the  occurrence  of  conjugation  of  two  such  individuals  by  their 
narrow  ends  to  form  a  zygote.  Further  observations,  however,  here  are 
necessary,  and  nothing  is  known  of  the  further  history  of  the  parasite 
within  the  insect  except  that  the  eggs  in  the  ovary  may  become  infected, 
so  that  insects  developed  from  these  can  carry  infection  to  animals. 
Frequently  when  an  animal  has  passed  through  an  attack  of  a  piro- 
plasmosis  it  is  immune  to  the  disease,  and  with  regard  to  this  immunity 
in  certain  cases  very  interesting  facts  have  been  observed.  For  instance, 
the  condition  may  not  be  associated  with  the  disappearance  of  the 
parasite  from  the  blood  of  the  immune  animal,  and  the  latter  may  thus 
be  a  source  of  danger  to  other  non-immune  animals  with  which  ticks 
harboured  by  it  may  come  in  contact. 

The  following  are  the  chief  piroplasmata  causing  disease  in  animals  : — 
(1)  Piroplasma  bigeminum.  This  was  first  described  by  Theobald  Smith 
and  is  the  cause  of  Texas  or  red-water  fever,  a  febrile  condition  associated 
with  h'semoglobinuria,  which  occurs  in  the  Southern  States  of  America, 
the  Argentine,  South  and  Central  Africa,  Algeria,  various  parts  of 
Northern  Europe,  and  in  Australia.  The  organism  gets  its  name  of 
bigeminum  from  the  fact  that  it  is  often  present  in  the  red  cells  in  pairs, 
which  may  be  attached  to  one  another  by  a  fine  thread  of  protoplasm  ; 
this  probably  results  from  the  complete  separation  of  two  individuals 
being  delayed  after  division  has  occurred.  Infection  is  here  spread  by 
the  tick  boophilus  bovis,  and  some  of  the  characteristics  of  the  disease 
epidemiologically  are  explained  by  the  fact  that  this  insect  goes  through 
all  its  moultings  on  the  same  individual  host.  (2)  Piroplasma  parvum. 
This  organism  was  discovered  by  Theiler  in  the  blood  of  cattle  suffering 
from  African  East  Coast  fever,  a  disease  closely  resembling  Texas  fever, 
which  prevails  endemically  in  a  narrow  strip  along  a  long  extent  of  the  east 
coast,  and  which  occurs  epidemically  inland.  As  its  designation  implies, 
the  organism  is  small,  and  it  is  also  attenuated.  Its  insect  host  is  the  tick 
rhipicephalus  appendiculatus,  and  it  may  be  noted  that  this  tick  drops 
off  the  animal  on  which  it  may  be  feeding  when  it  is  about  to  go  through 
one  of  its  several  moultings.  It  can  thus  carry  an  infection  much  more 
quickly  and  widely  through  a  herd  than  can  the  carrier  of  ordinary  red- 
water  fever.  It  may  be  said  that  in  England  there  occurs  a  red-water 
fever  also  associated  with  the  presence  of  a  piroplasm  in  the  blood,  but 
the  relationship  of  this  organism  to  the  other  varieties  has  not  yet  been 
fully  worked  out.  (3)  Piroplasma  equi.  This  organism  gives  rise  to 
biliary  fever  in  horses,  another  South  African  disease,  and  it  is  carried 


570  PIROPLASMOSIS 

by  the  tick  rhipicephalus  evertsii.  In  this  disease  Theiler  made  the 
interesting  observation  that  when  the  blood  of  a  donkey  which  had 
recovered  from  the  disease  was  injected  into  a  horse  the  latter  suffered  a 
slight  illness  only,  although  the  organisms  were  present  in  the  blood 
injected.  Such  a  fact  is  of  importance,  as  attenuation  of  virulence  in 
pathogenic  protozoa  seems,  so  far  as  our  present  knowledge  goes,  a  not 
very  common  event.  (4)  Piroplasma  canis.  This  causes  a  piroplasmosis 
occurring  in  dogs. 

With  regard  to  the  pathology  of  infection  by  piroplasmata  we  know 
nothing.  The  diseases  are  often  extremely  fatal,  carrying  off  nearly 
every  individual  attacked,  but  we  do  not  know  the  nature  of  the 
changes  originated. 


BIBLIOGRAPHY. 

GENERAL  TEXT- BOOKS. — In  English  the  student  may  consult  the  follow- 
ing :  "Micro-organisms  and  Disease,"  E.  Klein,  3rd  ed.,  London,  1896. 
"Bacteriology  and  Infective  Diseases,"  Edgar  M.  Crookshank,  London, 
1898.  "A  Manual  of  Bacteriology,"  George  M.  Sternberg,  New  York, 
1st  ed.  1893,  2nd  ed.  1896  (this  book  contains  a  full  bibliography).  ' '  Text- 
Book  upon  the  Pathogenic  Bacteria,"  Joseph  M'Farland,  London,  5th  ed., 
1906.  "Practical  Bacteriology,"  A.  A.  Kanthack  and  J.  H.  Drysdale, 
London,  1895.  "  Bacteria  and  their  Products,"  G.  S.  Woodhead,  London, 
1891.  "Bacteriological  Technique,"  Eyre,  London,  1902.  The  articles 
on  bacteriological  subjects  in  Clifford  Allbutt's  "System  of  Medicine," 
London,  are  of  the  highest  excellence,  and  have  full  bibliographies 
appended.  For  the  hygienic  aspects  of  bacteriology  see  "System  of 
Hygiene,"  Stevenson  and  Murphy,  London,  1892-94. 


by  Garnsey  and  Balfour,  Oxford,  1887  ;  Sachs,  "Text-Book  of  Botany," 
ii.,  transl.  by  Garnsey  and  Balfour,  Oxford,  1887. 

In  German:  "Die  Mikroorganismen,"  by  Dr.  C.  Fliigge,  3rd  ed., 
Leipzig,  1896.  "  Lehrbuch  der  pathologischen  Mykologie,"  by  Baum- 
garten,  Braunschweig,*  1890.  "Handbuch  der  pathogenen  Mikro- 
organismen," Kolle  and  Wassermann,  Fischer,  Jena,  1904. 

In  French  :  Roger,  "  Les  maladies  infectieuses,"  Paris,  1902. 

PERIODICALS. — For  references  to  current  work  see  (1)  Centralbl.  f. 
Bdkteriol.  u.  Parasitenk.,  Jena.  This  publication  commenced  in  1887. 
Two  volumes  are  issued  yearly.  In  1895  it  was  divided  into  two  parts. 
Abtheilung  I.  deals  with  Medizinisch-hygienische  Bakteriologie  und 
thierische  Parasitenkunde.  The  volumes  of  this  part  are  numbered  con- 
secutively with  those  of  the  former  series,  the  first  issued  thus  being  vol. 
xvii.  Commencing  in  1902  with  volume  xxxi.,  each  volume  of  Ab- 
theilung I.  was  further  divided  into  two  parts,  one  consisting  of  Originale 
the  other  of  Referate.  Abtheilung  II.  deals  with  Allgemeine  landwirt- 
schaftlich-technologische  Bakteriologie,  Garungs-pliysiologie  und  Pflanzen- 
pathologie.  The  first  volume  is  entitled  Zweite  Abtheilung,  Bd.  I.  It 
contains  original  articles,  Referate,  etc.  (2)  Bull,  de  I'lnsL  Pasteur, 
Paris,  Masson.  Besides  bacteriological  abstracts  this  journal  contains 
many  valuable  reviews  and  analyses  relating  to  protozoology.  (3)  "  Ergeb- 
nisse  der  allgemeinen  Pathologie,"  Lubarsch  and  Ostertag,  Wiesbaden, 
Bergmann.  This  from  time  to  time  contains  valuable  critical  reviews. 

The  most  complete  account  of  the  work  of  the  year  is  found  in  the 
Jahresb.  ii.  d,  Fortschr.  .  .  .  d.  path.  Mikroorganismen,  conducted  by 

571 


572  BIBLIOGRAPHY 

Baumgarten,  and  published  in  Braunschweig.  This  publication  com- 
menced in  1887.  Its  disadvantage  is  that  the  volume  for  any  year  does 
not  usually  appear  till  two  years  later. 

Bacteriology  is  also  dealt  with  in  the  Index  Medicus.  For  valuable 
lists  of  papers  by  particular  authors  see  Royal  Society  Catalogue  of 
Scientific  Papers  and  Internat.  Cat.  Sc.  Lit.  (Section,  Bacteriology). 

The  chief  bacteriological  periodicals  are  the  Journ.  Path,  and  Bacterial., 
Cambridge,  edited  by  G.  Sims  Woodhead  ;  the  Ztschr.  f.  Hyg.  u.  Infec- 
tionskrankh.,  Leipzig,  edited  by  Koch  and  Fliigge,  and  the  Ann.  de  I'lnst. 
Pasteur,  Paris^  edited  by  Duclaux  ;  Journ.  Exper.  Med.,  New  York, 
edited  by  Flexner  ;  Journ.  Hyg.,  Cambridge,  edited  by  Nuttall ;  Journ. 
Med.  Research,  Boston,  edited  by  Ernst ;  Journ.  Infect.  Diseases,  Chicago, 
edited  by  Hektoen  ;  Journ.  of  Royal  Army  Medical  Corps,  edited  by 
Bruce. 

Valuable  papers  also  from  time  to  time  appear  in  the  Lancet,  Brit. 
Med.  Journ.,  Deutsche  med.  Wchnschr.,  Berl.  klin.  Wchnschr.,  Semaine 
med.,  Arch.  f.  Hyg.,  Arch.  f.  exper.  Path.  u.  Pharmakol.  Besides  these 
periodicals  the  student  may  have  to  consult  the  Reports  of  the  Med.  Off. 
of  the  Local  Government  Board,  which  contain  the  reports  of  the  medical 
officers,  also  the  Proc.  Roy.  Soc.  London,  the  Compt.  rend.  Acad.  d.  sc. 
Paris,  the  Compt.  rend.  Soc.  de  biol.  Paris,  and  the  Arb.  a.  d.  k.  Gsndht- 
samte.  (the  first  two  volumes  of  the  last  were  denominated  Mittheilungen). 


CHAPTER  I. — GENERAL  MORPHOLOGY  AND  BIOLOGY. 

Consult  here  especially  Fliigge,  "Die  Mikroorganismen. "  De  Bary, 
"Bacteria,"  translated  by  Garnsey  and  Bayley  Balfour,  Oxford,  1887. 
Zopf,  "  Zur  Morphologie  der  Spaltpflanzen,"  Leipzig,  1882;  "Beitr.  z. 
Physiologic  uud  Morphologie  niederer  Organismen,"  5th  ed.,  Leipzig, 
1895.  Cohn,  Beitr.  2.  Biol.  d.  Pflanz.,  Bresl.  (1876),  ii.  v.  Nageli, 
"Die  niederen  Pilze,"  Munich,  1877;  "  Untersuchungen  iiber  niedere 
Pilze,"  Munich,  1882.  Migula,  "System  der  Bakterien,"  Jena,  1897. 
Duclaux,  "Traite  de  microbiologie, "  Paris,  1898-99.  For  general 
morphological  relations  see  Marshall  Ward,  art.  "Schizomycetes," 
Ency.  Brit.,  9th  ed.  xxi.  398;  xxvi.  51.  Engler  and  Prantl,  "Die 
naturlichen  Pflanzenfamilien,"  Lieferung,  129.  "  Schizophy  ta  "  (by  W. 
Migula).  STRUCTURE  OF  BACTERIAL  CELL. — Biitschli,  "Uber  den  Bau 
der  Bakterien,"  Leipzig,  1890;  "Weitere  Ausfuhrungen  iiber  den  Bau 
der  Cyanophyceen  und  Bakterien,"  Leipzig,  1896.  Fischer,  op.  cit.  in 
text.  Buchner,  Longard  and  Riedlin,  Centralbl.  f.  Bakteriol.  u.  Para- 
sitenk. ii.  1.  Ernst,  Ztschr.  f.  Hyg.  v.  428  ;  Babes,  ibid.  v.  173. 
Neisser,  ibid.  iv.  165.  MOTILITY. — Klein,  Biitschli,  Fischer,  Cohn, 
loc.  cit.  Loffler,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  vi.  209  ;  vii.  625. 
PIGMENTS. — Zopf,  loc.  cit.  ;  Galeotti,  Ref.  in  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.  xiv.  696.  Babes,  Ztschr.  f.  Hyg.  xx.  3.  SPORULATION. — 
Prazmowski,  Biol.  Centralbl.  viii.  301.  A.  Koch,  Botan.  Ztg.  (1888), 
Nos.  18-22.  Buchner,  Sitzungsb.  d.  math.-phys.  Cl.  d.  k.  layer.  Akad. 
d.  Wissensch.  zu  Munchen,  7th  Feb.  1880.  R.  Koch,  Mitth.  a.  d.  k. 
Gsndhtsamte.  i.  65.  CHEMICAL  STRUCTURE  OF  BACTERIA. — Nencki, 
Ber.  d.  deutsch.  chem.  Gesellsch.  (1884),  xvii.  2605.  Cramer,  Arch.  f. 
Hyg.  xvi.  154.  Buchner,  Berl.  klin.  Wchnschr.  (1890),  673,  1084  ;  vide 
Fliigge,  op.  cit.  CLASSIFICATION  OF  BACTERIA. — For  general  review 
see  Marshall  Ward,  Ann.  of  Botany,  vi.  103  ;  Migula,  loc.  cit.  supra. 


BIBLIOGRAPHY  573 

FOOD  OF  BACTERIA. — Nageli,  Cohn,  op.  cit.  Pasteur,  "  Etudes  sur  la 
biere,"  1876.  Hueppe,  Mitth.  a.  d.  k.  Gsndhtsamte.  ii.  309.  RELATIONS 
TO  OXYGEN. — Pasteur,  Compt.  rend.  Acad.  d.  sc.  Hi.  344,  1142  ;  Kitasato 
and  Weyl,  Ztschr.  f.  Hyg.  viii.  41,  404  ;  ix.  97.  TEMPERATURE. — Vide 
Fliigge,  op.  cit.  ;  for  thermophilic  bacteria,  Rabinowitsch,  Ztschr.  f. 
Hyg.  xx.  154  ;  Macfadyen  and  Blaxall,  Journ.  Path,  and  Bacteriol.  iii. 
87.  ACTION  OF  BACTERIAL  FERMENTS. — Salkowski,  Ztschr.  f.  BioL, 
N.F.,  vii.  92  ;  Pasteur  and  Joubert,  Compt.  rend.  Acad.  d.  sc.  Ixxxiii.  5 ; 
Sheridan  Lea,  Journ.  Physiol.  vi.  136  ;  Beijerinck,  Centralbl.  /.  Bak- 
teriol.  u.  Parasitenk.,  Abth.  II.  i.  221  ;  E.  Fischer,  Ber.  d.  deutsch.  chem. 
Gesellsch.  xxviii.  1430 ;  Liborius,  Ztschr.  f.  Hyg.  i.  115  ;  see  also 
Pasteur,  "Royal  Society  Catalogue  of  Scientific  Papers."  VARIABILITY. 
—Cohn,  Nageli,  Fliigge,  op.  cit.  Winogradski,  "  Beitr.  z.  Morph.  u. 
Physiol.  d.  Bakt.,"  Leipzig,  1888  ;  Ray  Lankester,  Quart.  Journ.  Micr. 
Sc.,  N.S.  (1873),  xiii.  408  ;  (1876),  xvi.  27,  278.  NITRIFYING  ORGANISMS. 
—Winogradski,  Ann.  de  Vlnst.  Pasteur,  iv.  213,  257,  760  ;  v.  92,  577. 
Maze,  ibid.  xi.  44  ;  xii.  1,  263. 


CHAPTER  II. — METHODS  OF  CULTIVATION  OF  BACTERIA. 

For  GENERAL  PRINCIPLES. — Pasteur,  Compt.  rend.  Acad.  d.  sc.  1. 
303  ;  li.  348,  675  ;  Ann.  de  chem.  Ixiii.  5  ;  Tyndall,  "Floating  Matter 
of  the  Air  in  relation  to  Putrefaction  and  Infection,"  London,  1881  ; 
H.  C.  Bastian,  "The  Beginnings  of  Life,"  London,  1872.  METHODS  OF 
STERILISATION. — R.  Koch,  Gatf'ky,  and  Loftier,  Mitth.  a.  d.  k.  Gsndht- 
samte. i.  322  ;  Koch  and  Wolff  hiigel,  ibid.  i.  301.  CULTURE  MEDIA. 
— See  text -books,  especially  Kanthack  and  Drysdale,  Evre  ;  Pasteur, 
"Etudes  sur  la  biere,"  Paris,  1876  ;  R.  Koch,  Mitth.  a." d.  k.  Gsndht- 
samte. i.  1  ;  Roux  et  Nocard,  Ann.  de  I'Inst.  Pasteur,  i.  1  ;  Roux,  ibid. 
ii.  28  ;  Marmorek,  ibid.  ix.  593  ;  Kitasato  and  Weyl,  op.  cit.  supra  ;  P. 
and  Mrs.  Percy  Frankland,  "Micro-organisms  in  Water,"  London,  1894. 
Fuller,  Rep.  Amer.  Pub.  Health  Ass.  xx.  381.  Theobald  Smith, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  vii.  502  ;  xiv.  864.  Durham, 
Brit.  Med.  Journ.  (1898),  i.  1387.  "Report  of  American  Committee  on 
Bacteriological  Methods,-"  Concord,  1898.  MacConkey,  Thompson- 
Yates  and  Johnston  Lab.  Rep.  vol.  iii.  pt.  iii.  151  ;  vol.  iv.  pt.  i.  p.  151  ; 
Journ.  Hyg.  v.  333.  Drigalski  and  Conradi,  Ztschr.  f.  Hyg.  xxxix. 
283. 

CHAPTER  III. — MICROSCOPIC  METHODS,  ETC. 

Consult  text-books,  especially  Klein,  Kanthack  and  Drysdale,  Hueppe, 
Giinther,  Heim  ;  also  Bolles  Lee,  "The  Microtomist's  Vademecum," 
London,  1905  (this  is  the  most  complete  treatise  on  the  subject). 
Rawitz,  op.  cit.  in  text ;  Koch,  Mitth.  a.  d.  k.  Gsndhtsamte.  i.  1  ; 
Ehrlich,  Ztschr.  f.  klin.  Med.  i.  553  ;  ii.  710.  Gram,  Fortschr.  d. 
Med.  (1884),  ii.  No.  6  ;  Nicholle,  Ann.  de  I'Inst.  Pasteur,  ix.  666  ; 
Ktihne,  "  Praktische  Anleitung  zum  mikroscopischen  Nachweis  der 
Bakterien  im  tierischen  Gewebe,"  Leipzig,  1888  ;  van  Ermengem,  ref. 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xv.  969  ;  Richard  Muir,  Journ. 
Path,  and  Bacteriol.  v.  374  ;  Mann,  "  Physiological  Histology," 
Oxford,  1902.  For  Romanowsky  methods  see  Jenner,  Lancet  (1899), 


574  BIBLIOGRAPHY 

i.  370  ;  Leishman,  Brit.  Med.  Journ.  (1901),  i.  635  ;  (1902),  ii.  757  ; 
Journ.  of  Roy.  Army  Med.  Corps  (1904),  ii.  669  ;  Geimsa,  Deutsche 
Med.  Wchnschr.  (1905),  1026;  Ann.  de  I'lnst.  Pasteur,  xix.  346; 
MacNeal,  Journ.  Inf.  Dis.  iii.  412;  Wright,  J.  H.,  Journ.  Med.  Res. 
vii.  138. 

AGGLUTINATION. — Delepine,  Brit.  Med.  Journ.  (1897),  ii.  529,  967. 
Widal  and  Sicard,  Ann.  de  I'lnst.  Pasteur,  xi.  353.  Wright,  Brit.  Med. 
Journ.  (1897),  i.  139  ;  (1898),  i.  355. 


CHAPTER  IV. — BACTERIA  OF  AIR,  SOIL,  WATER.     ANTISEPTICS. 

AIR,  SOIL,  AND  WATER.  —  Petri,  Ztschr.  f.  Hyg.  iii.  1  ;  vi.  233. 
Fliigge,  ibid.  xxv.  179.  Sticher,  ibid.  xxx.  163.  Weyl,  "  Handbuch 
der  Hygiene,"  Jena,  1896,  et  seq.  Houston,  Rep.  Med.  Off.  Local  Gov. 
Bd.  xxvii.  (1897-98)  251  ;  xxviii.  (1898-99)  413,  439,  467  ;  xxix.  (1899- 
1900)  458,  489.  Sidney  Martin,  ibid.  xxvi.  (1896-97)  231  ;  xxvii. 
(1897-98)  308;  xxviii.  (1898-99)  382.  Horrocks,  "Bacteriological 
Examination  of  Water,"  London,  1901.  Percy  and  G.  C.  Frankland, 
"Micro-organisms  in  Water,"  London,  1894.  Dibdin,  "  Purification  of 
Sewage  and  Water,"  London,  1897.  Ann.  Rep.  Bd.  Health,  Mass., 
Boston,  1890,  et  seq.  Savage,  "The  Bacteriol.  Exam,  of  Water  Supplies, " 
London,  1906.  Lewis,  Rideal,  and  Walker,  Journ.  Roy.  San.  Inst. 
(1903),  xxiv.  424. 

ANTISEPTICS.— R.  Koch,  Mitth.  a.  d.  k.  Gsndhtsamte.  i.  234.  Behriug, 
Ztschr.  f.  Hyg.  ix.  395.  Ritchie,  Trans.  Path.  Soc.  London,  1.  256. 
Rideal,  "Disinfection  and  Disinfectants,"  London,  1898. 


CHAPTER  V.— RELATIONS  OF  BACTERIA  TO  DISEASE,  ETC. 

As  the  observations  on  which  this  chapter  is  based  are  scattered 
through  the  rest  of  the  book,  the  references  to  them  will  be  found  under 
the  different  diseases. 


CHAPTER  VI. — INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS. 

Ogston,  Brit.  Med.  Journ.  (1881),  i.  369.  Rosenbach,  "  Mikro- 
organismen  bei  den  Wundinfectionskrankheiten  des  Menschen,"  Wies- 
baden, 1884.  Passet,  Fortschr.  d.  Med.  (1885),  Nos.  2  and  3.  W. 
Watson  Cheyne,  "Suppuration  and  Septic  Diseases,"  Edinburgh,  1889. 
Grawitz,  Virclwws  Archiv,  cxvi.  116  ;  Deutsche  med.  Wchnschr.  (1889), 
No.  23.  Steinhaus,  Ztschr.  f.  Hyg.  v.  518  (micrococcus  tetragenus) ; 
'•'Die  Aetiologie  der  acuten  Eiterung,"  Leipzig,  1889.  Christmas- 
Dirckinck-Holmfeld,  "  Recherches  experimentales  sur  la  suppuration," 
Paris,  1888.  Muir,  Journ.  Path,  and  Bacteriol.  vii.  161  ;  Trans.  Path. 
Soc.  London,  1902.  Garre,  Fortschr.  d.  Med.  (1885),  No.  6.  Marmorek, 
Ann.  de  I'lnst.  Pasteur,  ix.  593.  Petruschky,  Ztschr.  f.  Hyg.  xvii.  59  ; 
xviii.  413  ;  xxiii.  142  ;  (with  Koch,  xxiii.  477).  Liibbert,  "Biologische 
Spaltpilzuntersuchung,"  Wiirzburg,  1886.  Krause,  Fortschr.  d.  Med. 
(1884),  Nos.  7  and  8.  Ribbert,  Fortschr.  d.  Med.  (1886),  No.  1.  Widal 
and  Besancon,  Ann.  de  I'lnst.  Pasteur,  ix.  104.  v.  Lingelsheim,  Ztschr. 


BIBLIOGRAPHY  575 

/.  Hyg.  x.  331  ;  xii.  308.  Behring,  Oentralbl.  f.  Bakteriol.  u.  Parasitenk. 
xii.  192.  Thoiuot  et  Masselin,  Rev.  de  m<*d.  (1894),  449.  Ortli  and 
Wyssokowitsch,  Centralbl.  f.  d.  med.  Wissensch.  (1885),  577.  Netter, 
Arch,  de  physiol.  norm,  et  path.  (1886),  106.  Weicliselbaura,  Wien.  med. 
Wchnsehr.  (1885),  No.  41  ;  (1888),  Nos.  28-32  ;  Centralbl.  f.  BaUeriol.  u. 
Parasitenk.  ii.  209  ;  Beitr.  z.  path.  Anat.  u.  z.  allg.  Path.  iv.  127. 
Becker,  Deutsche  med.  Wchnsehr.  (1883),  No.  46.  Lannelongue  et 
Achard,  Ann.  de  I'lnst.  Pasteur,  v.  209.  Fehleisen,  "Die  Aetiologie 
des  Erysipels,"  Berlin,  1883.  Welch,  Am.  Med.  Journ.  Sc.  (1891),  439. 
Lemoine,  Ann.  de  I'lnst.  Pasteur,  ix.  877.  Kurth,  Arb.  a.  d.  k. 
Gsndhtsamte.  vii.  389.  Knorr,  Ztschr.  f.  Hyg.  xiii.  427.  Bulloch, 
Lancet  (1896),  i.  982,  1216.  Bordet,  Ann.  de  I'lnst.  Pasteur,  xi.  177. 
Booker  (streptococcus  enteritis),  Johns  Hopkins  Hosp.  Rep.  vi.  159. 
Hirsch,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxii.  369.  Libman,  ibid. 
xxii.  376.  Wright  and  Douglas,  Proc.  Roy.  Soc.  Lond.  Ixxiv.  147. 
Wright,  Clinical  Journal  (1906),  May  16. 

Streptococci. — Hiss,  Journ.  Exper.  Med.  vi.  317.  Schottmuller, 
Munchen.  med.  Wchnsehr.  (1903),  849.  Gordon,  Reports  Med.  Officer 
Local  Gov.  Board  (1905),  388  ;  Lancet  (1905),  ii.  1400.  Andrewes  and 
Horder,  Lancet  (1906),  ii.  Ruediger,  Journ.  Infect.  Dis.  iii.  755. 
Besredka,  Bull,  de  I'lnst.  Pasteur,  iii. 

Conjunctivitis. — Morax,  Ann.  de  I'lnst.  Pasteur  (1896),  x.  337.  Eyre, 
Journ.  Path.  and.  Bacteriol.  vi.  1.  Miiller,  Wien.  med.  Wchnsehr. 
1897  ;  Inglis  Pollock,  Trans.  Ophthalm.  Soc.  1905  ;  Axenfeld,  in  Lubarsch 
and  Ostertag,  "Ergebnisse  der  allgem.  Pathol.  u.  Path.  Anat.,"  1901  ; 
"Die  Bakteriologie  in  der  Augenheilkunde,"  1907  (full  references). 

Acute  Rheumatism. — Triboulet  and  Cayon,  Bull.  Soc.  med.  d.  hop.  de 
Paris  (1898),  93.  Westphal,  Wassermann,  and  Malkoff,  Berl.  klin. 
Wchnsehr.  (1899),  638.  Poynton  and  Paine,  Lancet  (1900),  ii.  861,  932 
(full  references).  Trans.  Path.  Soc.  Lond.  (1902),  liii.  221  ;  Lancet, 
December  1905.  Beaton  and  Walker,  Brit.  Med.  Journ.  (1903),  i.  237. 
Shaw,  Journ.  Path,  and  Bacteriol.  (1903),  ix.  158.  Beattie,  ibid.  ix.  272  ; 
Journ.  Med.  Research,  xiv.  399  ;  Journ.  Exper.  Med.  ix.  186.  Cole, 
Journ.  Infect.  Dis.  i.  714. 


CHAPTER  VII.  —  INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS, 
CONTINUED  :  ACUTE  PNEUMONIAS,  EPIDEMIC  CEREBRO  -  SPINAL 
MENINGITIS. 

Friedlander,  Fortschr.  d.  Med.  i.  No.  22  ;  ii.  287  ;  Virchow's  Archiv, 
Ixxxvii.  319.  A.  Fraenkel,  Ztschr.  f.  klin.  Med.  (1886),  401.  Salvioli 
and  Zaslein,  Centralbl.  f.  d.  med.  Wissensch.  (1883),  721.  Ziehl,  ibid. 
(1883),  433  ;  (1884),  97.  Klein,  ibid.  (1884),  529.  Jiirgensen,  Berl.  klin. 
Wchnsehr.  (1884),  270.  Seibert,  ibid.  (1884),  272,  292.  Senger,  Arch, 
f.  exper.  Path.  u.  Pharmakol.  (1886),  389.  Weichselbaum,  Wien.  med. 
Wchnsehr.  xxxvi.  1301,  1339,  1367  ;  Monatschr.  f.  Ohrenh.  (1888),  Nos. 
8  and  9  ;  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  v.  33.  Gamaleia,  Ann. 
de  I'lnst.  Pasteur,  ii.  440.  Guarnieri,  Atti  d.  r.  Accad.  med.  di  Roma 
(1888),  ser.  ii.  iv.  Kruse  and  Pansini,  Ztschr.  f.  Hyg.  xi.  279.  E. 
Fraenkel  and  Reiche,  Ztschr.  f.  klin.  Med.  xxv.  230.  Sanarelli,  Centralbl. 
f.  Bakteriol.  u.  Parasitenk.  x.  817.  Lannelongue,  Gaz.  d.  hop.  (1891), 
379.  Netter,  Bull,  et  mtm.  Soc.  med.  d.  hop.  de  Paris  (1889) ;  Compt. 
rend.  Acad.  d.  sc.  (1890) ;  Compt.  rend.  Soc.  de.  biol.  Ixxxvii.  34. 


576  BIBLIOGRAPHY 

G.  and  F.  Klemperer,  BerL  klin.  Wchnschr.  (1891),  893,  869.  Fo&  and 
Bordoni-Uffreduzzi,  Deutsche  med.  Wchnschr.  (1886),  No.  33.  Emmerich, 
Munchen.  med.  Wchnschr.  (1891),  No.  32.  Issaeff,  Ann.  de  I'lnst. 
Pasteur,  vii.  260.  Grimbert,  Ann.  de  TInst.  Pasteur,  xi.  840.  Wash- 
bourn,  Brit.  Med.  Journ.  (1897),  i.  510  ;  (1897),  ii.  1849.  Eyre  and 
Washbourn,  Journ.  Path,  and  Bacterial,  iv.  394 ;  v.  13.  See  also 
Brit.  Med.  Journ.  (1901),  ii.  760;  Neufeld  and  Rimpau,  Ztschr.  f. 
Hyg.  Ii.  283. 

Meningitis.—  Weichselbaum,  Fortschr.  d.  Med.  (1887),  v.  573,  620. 
Jaeger,  Ztschr.  f.  Hyg.  xix.  351.  Councilman,  Mallory,  and  Wright, 
"Epidemic  Cerebro-spinal  Meningitis,"  Rep.  Bd.  Health  Mass.,  Boston, 
1898  (full  references).  Gwyn,  Johns  Hopkins  Hasp.  Bull.  (1899),  109. 
v.  Lingelsheim,  Klin.  Jahrb.  xv.  373.  Kolle  and  Wassermann,  ibid. 
p.  507.  Kutscher,  Deutsche  med.  Wchnschr.  (1906),  1071.  Bettencourt 
and  Franca,  Ztschr.  f.  Hyg.  xlvi.  463.  Durham,  Journ.  Infect.  Dis. 
Suppl.  No.  2,  p.  10.  Goodwin  and  von  Sholly,  ibid.  p.  21.  Arkwright, 
Journ.  of  Hyg.  vii.  145.  Flexner,  Journ.  Exper.  Med.  ix.  105.  Van- 
steenberghe  et  Grysez,  Ann.  de  I'lnst.  Pasteur,  xx.  69. 


CHAPTER  VIII.—  GONORRHOEA,  SOFT  SORE,  SYPHILIS. 

GONORRHOEA. — Neisser,  Centralbl.  f.  d.  med.  Wissensch.  (1879),  497  ; 
Deutsche  med.  Wchnschr.  (1882),  279  ;  (1894),  335.  Bumm,  "  Der  Mikro- 
organismus  der  gonorrhoischen  Schleimhauterkrankungen,"  Wiesbaden, 
1885,  2nd  ed.  1887  ;  Munchen.  med.  Wchnschr.  (1886),  No.  27  ;  (1891), 
Nos.  50  and  51  ;  Centralbl.  f.  Gyndk.  (1891),  No.  22  ;  Wien.  med.  Presse 
(1891),  No.  24.  Bockhart,  Monatsh.  f.  prakt.  Dermal.  (1886),  v.  No.  4  ; 
(1887),  vi.  No.  19.  Steinschneider,  Berl.  klin.  Wchnschr.  (1890),  No. 
24  ;  (1893),  No.  29  ;  Verhandl.  d.  deutsche  dernmt.  Gesellsch.  I.  Congress, 
Wien  (1889),  159.  Wertheim,  Wien.  klin.  Wchnschr.  (1890),  25  ; 
Deutsche  med.  Wchnschr.  (1891),  No.  50  ;  Arch.  f.  Gyndk.  xli.  Heft  1  ; 
Centralbl.  f.  Gyndk.  (1891),  No.  24;  (1892),  No.  20;  Wien.  klin. 
Wchnschr.  (1894),  441.  Gerhardt,  Charity-Ann.  (1889),  241.  Leyden, 
Ztschr.  f.  klin.  Med.  xxi.  607  ;  Deutsche  med.  Wchnschr.  (1893),  909. 
Bordoni-Uffreduzzi,  ibid.  (1894),  484.  Councilman,  Am.  Journ.  Med. 
Sc.  cvi.  277.  Finger,  Ghon,  and  Schlagenhaufer,  Arch.  f.  Dermat.  u. 
Syph.  xxviii.  3,  276.  Lang,  ibid;  (1892),  1007  ;  Wien.  med.  Wchnschr. 
(1891),  No.  7;  "Der  Venerische  Katarrh,  dessen  Pathologic  und 
Therapie,"  Wiesbaden,  1893.  Klein,  Monatschr.  f.  Geburtsh.  u.  Gynaek. 
(1895),  33.  Michaelis,  Ztschr.  f.  klin.  Med.  xxix.  556.  Heiman,  New 
York  Med.  Rec.  (1895),  769  ;  (1896),  Dec.  19.  Foulerton,  Trans.  Brit. 
Inst.  Preven.  Med.  i.  40.  De  Christmas,  Ann.  de  I'lnst.  Pasteur,  xi. 
609.  Nicolaysen,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxii.  305. 
Rendu,  Berl.  klin.  Wchnschr.  (1898),  431.  Wassermann,  Ztschr.  f. 
Hyg.  xxvii.  298  ;  Munchen.  med.  Wchnschr.  (1901),  No.  8.  Lenhartz, 
Berl.  klin.  Wchnschr.  (1897),  1138.  Thayer  and  Lazear,  Journ.  Exper. 
Med.  iv.  81.  Konig,  Berl.  klin.  Wchnschr.  (1900),  No.  47.  De  Christmas, 
Ann.  de  I'lnst.  Pasteur,  (1900),  xiv.  331.  Raskai,  Deutsche  med.  Wchnschr. 
(1901),  No.  1.  Jundell,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxix. 
224.  Colombini,  ibid.  xxiv.  955.  Bressel,  Munchen.  med.  Wchnschr. 
(1903),  No.  13.  Holler,  Arch.  f.  Dermat.  u.  Syph.  (1904),  Ixxi.  269. 
Wynn,  Lancet  (1905),' i.  352.  Prochaska,  Arch.  f.  klin.  Med.  Ixxxiii. 
Heft  1-2.  Strong,  Journ.  Am.  Med.  As.,  May  1904. 


BIBLIOGRAPHY  577 

SOFT  SORE. — Ducrey,  Monatsh.  f.  prakt.  Dermal,  ix.  221.  Krefting, 
Arch.f.  Dermat.  u.  Syph.  (1892),  263.  Jullien,  Journ.  d.  mal.  cutan.  et 
syph.  (1892),  330.  Unna,  Monatsh.  f.  prakt.  Dermat.  (1892),  475  ;  (1895), 
61.  Quinquand,  Semaine  'med.  (1892),  278.  Petersen,  Centralbl.  f. 
Bakteriol.  u.  Parasitenk.  xiii.  743  ;  Arch.  f.  Dermat.  u.  Syph.  (1894), 
419.  Audrey,  Monatsh.  f.  prakt.  Dermat.  (1895),  267.  Colombini, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxv.  254.  Nicolle,  Presse  medicale 
(1900),  304.  Bezan9on,  Griffon,  and  Le  Sourd,  Ann.  de  dermat.  et  de 
syphilolog.  (1901),  tome  ii.  1.  Lenglet,  ibid.  (1901),  tome  ii.  209.  Simon, 
Compt.  rend.  Soc.  Biol.  (1902),  547.  Tomasczewski,  Ztschr.  f.  Hyg. 
(1903),  Bd.  43,  p.  327.  Davis,  Journ.  of  Med.  Research  (1903),  ix.  401. 

SYPHILIS.  —  Lustgarten,  Wien.  med.  Wchnschr.  (1884),  No.  47. 
Doutrelepont  and  Schutz,  Deutsche  med.  Wchnschr.  (1885),  No.  19. 
Gottstein,  Fortschr.  d.  Med.  (1885),  No.  16.  De  Michele  and  Radice, 
Gior.  internaz.  di  sc.  med.  (1892),  535.  Sabouraud,  Ann.  de  I'Inst. 
Pasteur,  vi.  184.  Golasz,  Journ.  d.  mal.  cutan.  et  syph.  (1894),  170. 
Markuse,  Vrtljschr.  f.  Dermat.  u.  Syph.  (1883),  No.  3.  Van  Niessen, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxiii.  49.  Metchnikoff  and  Roux, 
Ann.  de  I  Inst.  Pasteur,  xvii.-xix.  Lassar,  Berl.  klin.  Wchnschr.  (1903), 
1189.  Neisser,  Deutsche  med.  Wchnschr.  (1904),  1369,  1431.  Schatidinn 
and  Hoffmann,  Arb.  a.  d.  kaiserl.  Gesundheitsamte  (1905),  Bd.  22  ; 
Deutsche  med.  Wchnschr.  (1905),  No.  18  ;  Berl.  klin.  Wchnschr.  (1905), 
Nos.  22,  23.  Schaudinn,  Deutsche  med.  Wchnschr.  (1905),  No.  22. 
Hoffmann,  Berl.  klin.  Wchnschr,  (1905),  No.  46  ;  "Selected  Essays  on 
Syphilis  and  Smallpox,"  New  Sydenham  Society,  1906.  Metchnikoff, 
La  Semaine  m<*d.  (1905),  234.  Levaditi,  ibid.  (1905),  247.  Siegel, 
Milnchen.  med.  Wchnschr.  (1905).  1321,  1384.  Herxheimer,  ibid.  (1905), 
1857.  Shennan,  Lancet  (1906),  i.  663,  746.  Maclennan,  Brit.  Med.  Journ. 
(1906),  i.  1090.  Levaditi,  Ann.  de  I  Inst.  Pasteur  (1906),  41. 


CHAPTER  IX.— TUBERCULOSIS. 

Klencke,  "  Untersuchungen  und  Erfuhrungen  im  Gebiet  der  Anatomic, 
etc.,"  Leipzig,  1843.  Villemin,  "De  la  virulence  et  de  la  specificite  de 
la  tuberculose,"  Paris,  1868.  Cohnheim  and  Fraenkel,  "  Experimentelle 
Untersuchungen  iiber  der  tJbertragbarkeit  der  Tuberculose  auf  Thiere." 
Cohnheim,  "Die  Tuberculose  vom  Standpunkt  der  Infectionslehre," 
1879.  Various  Authors,  "Discussion  sur  la  tuberculose,"  Bull.  Acad. 
de  med.  (1867),  xxxii.,  xxxiii.  Armanni,  "Novimento  med.-chir.," 
Naples,  1872.  Baumgarten,  "Lehrb.  d.  path.  Myk.,"  1890.  Straus, 
"La  tuberculose  et  son  bacille,"  Paris,  1895.  Koch,  Berl.  klin. 
Wchnschr.  (1882),  221  ;  Mitth.  a.  d.  k.  Gsndhtsamte.,  1884  ;  Deutsche 
med.  Wchnschr.  (1890),  No.  46a.  ;  (1891),  Nos.  3  arid  43  ;  (1897),  No.  14. 
Bulloch,  Lancet  (1901),  ii.  243.  Nocard,  "The  Animal  Tuberculoses," 
(trans.),  London,  1895.  Cornet,  Ztschr.  f.  Hyg.  v.  1.91.  Nocard  and 
Roux,  Ann.  de  I'Inst.  Pasteur,  i.  19.  Pawlowsky,  ibid.  ii.  303. 
Sander,  Arch.  f.  Hyg.  xvi.  238.'  Coppen  Jones,  Centralbl.  f.  Bakteriol. 
u.  Parasitenk.  xvii.  1.  Prudden  and  Hodenpyl,  New  York  Med.  Rec. 
(1891),  636.  Vissman,  Virchoiv's  Archiv,  cxxix.  163.  Straus  and 
Gamaleia,  Arch,  de  med.  exptr.  et  d'anat.  path.  iii.  No.  4.  Courmont, 
Semaine  med.  (1893),  53  ;  Revue  de  med.  (1891),  No.  10.  Hericourt  and 
Richet,  Bull.  med.  (1892),  741,  966.  Williams,  Lancet^  (1883),  i.  312. 
Pawlowsky,  Ann.  de  I'Inst.  Pasteur,  vi.  116.  Maffucci,  "Sull'  azione 
37 


578  BIBLIOGRAPHY 

tossica  del  prodotti  del  bacillo  della  tuberculosi "  ;  Centralbl.  f.  allg. 
Path.  u.  path.  Anat.  i.  404.  Kruse,  Beitr.  z.  path.  Anat.  u.  z.  allg. 
Path.  xii.  221.  Bellinger,  Miinchen.  med.  Wchnschr.  (1889),  No.  37  ; 
Verhandl.  d.  Gesellsch.  deutsch.  Naturf.  u.  Aertze  (1890),  ii.  187. 
Hofmann,  Wien.  med.  Wchnschr.  (1894),  No.  38.  Straus  and  Wiirtz, 
Gong.  p.  I'ttude  de  la  tuberculose,  Paris,  July  1888.  Gilbert  and  Roger, 
Mem.  Soe.  de  Uol.  (1891).  Diem,  Monatsh.  f.  prakt.  Thierh.  iii.  481. 
Weyl,  Deutsche  med.  Wchnschr.  (1891),  256.  Buchner,  CentralbL  f. 
Bakteriol.  u.  Parasitenk.  xi.  488.  Courmont  and  Dor,  Province  med. 
(1890),  No.  50.  Tizzoni  and  Centanni,  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.  xi.  82.  Ribbert,  Deutsche  med.  Wchnschr.  (1892),  353. 
Virchow,  ibid.  (1891),  131.  Hunter,  Brit.  Med.  Journ.  (1891),  July 
25.  Kiihne,  Ztschr.  f.  Biol.  xxix.  1  ;  xxx.  221.  Krehl,  Arch.  f.  exper. 
Path.  u.  Pharmakol.  xxxv.  222.  Krehl  and  Matthes,  ibid,  xxxvi.  437. 
Bang,  "La  lutte  contre  la  tuberculose  en  Danemark,"  Geneva,  1895. 
Maragliano,  "  Le  serum  antituberculeux  et  son  antitoxin,"  Paris,  1896  ; 
Berl.^klin.  Wchnschr.  (1896),  409,  437,  773.  Nocard,  Ann.  de  TInst. 
Pasteur,  xii.  561.  Stockman,  Brit.  Med.  Journ.  (1898),  ii.  681.  Mara- 
gliano, ref.  Brit.  Med.  Journ.,  Epitome  (1896),  i.  63.  Baumgarten  and 
Walz,  CentralbL  f.  Bakteriol.  u.  Parasitenk.  xxiii.  587.  Smith,  T., 
Journ.  Exper.  Med.  iii.  451.  Koch.  Brit.  Med.  Journ.  (1901),  ii.  189  ; 
Trans.  Internat.  Congr.  of  Tuberc.,  London,  1901.  Delepine,  Brit. 
Med.  Journ.  (1901),  ii.  1224.  Bataillon,  Dubard,  and  Terre  (fish 
tuberculosis),  Compt.  rend.  Soc.  de  biol.  1897,  446.  Dubard,  Rev.  de  la 
tubercul.  (1898),  13,  129.  Ravenel,  Med.  Bull.  Univ.  Pennsylvania,  May 
1902.  Koch,  'Deutsche  med.  Wchnschr.  (1902),  No.  48.  Koch,  Schutz, 
Neufeld,  and  Miessner,  Ztschr.  f.  Hyg.  51,  300.  De  Jong,  Centralbl.  f. 
Bakteriol.  u.  Parasitenk.  38  (Orig.),  146.  Ravenel,  Univ.  of  Pennsylvania 
Med.  Bulletin,  1902.  Kossel,  Weber,  and  Heuss,  Tuberkulosearbeiten 
aus  d.  Kaiserl.  Gsndhtsmte.,  Berlin,  1904-1905.  Weber  and  Tante,  ibid. 
1905.  Salmon  and  Smith,  "Tuberculosis,"  U.S.  Department  of  Agri- 
culture, Washington,  1904.  Wolbach  and  Ernst,  Journ.  Med.  Research, 
x.  313.  Interim  Reports  of  the  Royal  Commission  on  Tuberculosis, 
London,  1904,  1907.  Wright  and  Douglas,  Proc.  Roy.  Soc.  Lond.,  Ixxiv. 
159.  Wright,  Clinical  Journal,  Nov.  9,  1904;  ibid.,  May  15,  1906; 
Med.  Chir.  Trans.  Ixxxix.  (1905).  Wright  and  Reid,  Proc.  Roy.  Soc. 
Lond.  Ixxvii.  194,  211. 

Other  acid-fast  bacilli.—  Moeller,  Deutsche  med.  Wchnschr.  (1898),  376. 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxv.  369  ;  ibid.  xxx.  513.  Petri, 
Arb..  a.  d.  k.  Gsndhtsamte.  (1898),  1.  Rabinowitch,  Deutsche  med. 
Wchnschr.  (1897),  No.  26  ;  (1900),  No.  16  ;  Ztschr.  f.  Hyg.  xxvi.  90. 
Korn,  Arch.  f.  Hyg.  xxxvi.  57  ;  Centralbl.  f.  Bakteriol.  u.  Parasitenk. 
xxvii.  481.  Schulze,  Ztschr.  f.  Hyg.  xxxi.  153.  M.  Tobler,  ibid,  xxxvi. 
120.  Lubarsch,  ibid.  xxxi.  187.  Holscher,  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.  xxix.  425.  Potet,  "  Etude  sur  les  bacilles  dites  '  acido- 
philes,'"  Paris,  1902.  Abbott  and  Gildersleeve,  Pennsylv.  Med.  Bullet., 
June  1902.  Johne  and  Frothingham,  Deutsche  Ztschr.  f.  Thiermed.  (1895), 
438.  M'Fadyean,  Journ.  Compar.  Path.  xx.  (1907),  48. 


CHAPTER  X.— LEPROSY. 

Hansen,  Norsk.  Mag.  f.  Lcegevidensk.,  1874  ;   Virchow' s  Archiv,  Ixxix. 
32  ;  xc.  542  ;  ciii.  388  ;  Virchow's  Festschr.  (1892),  iii.     See  papers  by 


BIBLIOGRAPHY  579 

Neisser  and  Cornil  and  Suchard  in  "  Microparasites  in  Disease"  (New 
Sydenham  Soc.,  1886).  Hansen  and  Looft,  "Leprosy,"  Bristol,  1895. 
Doutrelepont  and  Wolters,  Arch.  f.  Dermat.  u.  Syph.  (1892),  55. 
Thoma,  Sitzungsb.  d.  Dorpater  Naturforsch.,  1889.  Unna,  Dermat. 
Stud.  Hamburg  (1887),  iv.  Bordoni-Uffreduzzi,  Ztschr.  f.  Hyg.  iii. 
178  ;  Berl.  klin.  Wchnschr.  (1885),  No.  11.  Arning  and  Nonne, 
Virchows  Archiv,  cxxxiv.  319.  Gairdner,  Brit.  Med.  Journ.  (1887), 
i.  1269.  Hutchinson,  Arch.  Surg.  (1889),  i.  V.  Torb'k,  "Summary  of 
Discussion  on  Leprosy  at  the  1st  Internat.  Congr.  for  Dermatol.  and 
Syph."  v.  Monatsh.  f.  prakt.  Dermat.  ix.  238.  Profeta,  Gior.  internaz. 
d.  sc.  med.  1889.  See  Journal  of  the  Leprosy  Investigation  Committee, 
1890-91.  Philippson,  Virchows  Archiv,  cxxxii.  529.  Danielssen, 
Monatsh.  f.  prakt.  Dermat.  (1891),  85,  142.  Wesener,  Centralbl.  f. 
Bakteriol.  u.  Parasitenk.  ii.  450  ;  Milnchen.  med.  Wchnschr.  (1887), 
No.  18.  Uhlenhuth  and  Westphal,  Centralbl.  f.  Bakteriol.  u.  Parasitenk. 
xxix.  233.  Dean,  Journ.  of  Hyg.  v.  99.  Babes  in  "  Erganzungsband  " 
of  Kolle  and  Wassermanns  Handbuch  der  Pathogenen  Mikroorganismen. 


CHAPTER  XL— GLANDERS— RHINOSCLEROMA. 

Loffler  and  Schultz,  Deutsche  med.  Wchnschr.  (1882),  No.  52.  Loffler, 
Mitth.  a.  d.  k.  Gsndhtsamte.  i.  134.  Weichselbaum,  Wien.  med. 
Wchnschr.  (1885),  Nos.  21-24.  Preusse,  Berl.  thierdrztl.  Wchnschr. 
(1889),  Nos.  3,  5,  11  ;  ibid.  (1894),  Nos.  39,  51.  Gamaleia,  Ann.  de 
Vlnst.  Pasteur,  iv.  103.  A.  Babes,  Arch,  de  me"d.  expe"r.  et  d'anat.  path. 
(1892),  450.  Straus,  Compt.  rend.  Acad.  d.  sc.  cviii.  530.  M'Fadyean 
and  Woodhead,  Rep.  National  Vet.  Assoc.  1888.  Baumgarten,  Centralbl. 
f.  Bakteriol.  u.  Parasitenk.  iii.  379.  Silviera,  Semaine  mdd.  (1891), 
No.  31.  Bonome,  Deutsche  med.  Wchnschr.  (1894),  703,  725,  744. 
Kalning,  Arch.  f.  Veterindrwissensch.  (St.  Petersburg),  i.  Apr.  May. 
Foth,'  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xvi.  508,  550.  M'Fadyean, 
Journ.  Comp.  Path,  and  Therap.,  1892,  1893,  1894.  Leclainche  and 
Montane,  Ann.  de  VInst.  Pasteur,  vii.  481.  Leo,  Ztschr.  f.  Hyg.  vii. 
505.  Marx,  Centralbl.  /.  Bakteriol.  u.  Parasitenk.  xxv.  275.  Mayer, 
ibid,  xviii.  673.  Bonome,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  Refer, 
xxxviii.  97.  Anderson,  Chalmers,  and  Buchanan,  Glasgoio  Med.  Journ. , 
Oct.  1905.  Nicolle,  Ann.  de  I' List  Pasteur,  xx.  623,  698,  801. 

RHINOSCLEROMA. — Frisch,  Wien.  med.  Wchnschr.  (1882),  No.  32. 
Cornil  and  Alvarez,  Arch,  de  physiol.  norm,  et  path.  (1895),  3rd  series, 
vi.  11.  Paltaiif  and  Eiselsberg,  Fortschr.  d.  Med.  (1886),  Nos.  19,  20. 
WoJ»witsch,  Centralbl.  f.  d.  med.  Wissensch.,  1886.  Dittrich,  Ztschr. 
f.  Heilk.  viii.  251.  Babes,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  ii. 
617.  Pawlowski,  ibid.  ix.  742;  "Sur  1'etiologie  et  la  pathologic  du 
rhinosclerome,"  Berlin,  1891.  Paltauf,  Wien.  med.  Wchnschr.  (1891), 
Nos.  52,  53  ;  (1892),  Nos.  1,  2.  Wilde,  Semaine  med.  (1896),  336. 
Klemperer  and  Scheier,  Ztschr.  f.  klin.  Med.  xlv.  Heft  1-2.  Lanzi, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  Refer,  xxxiv.  627.  Schablowski, 
ibid,  xxxviii.  714. 


CHAPTER  XII.— ACTINOMYCOSIS,  ETC. 

Bollinger,  Centralbl.  f.  d.  med.   Wissensch.,  1877.     J.  Israel,  Virchow's 
Archiv,  Ixxiv.  15  ;  Ixxviii.  421.     Ponfick,  Breslau.  aertzl.  Ztschr.,  1879  ; 


580 


BIBLIOGRAPHY 


"Die  Aktinomykose  des  Menschen,"  1882.  O.  Israel,  Virchows  Archiv 
xcvi.  175.  Chiari,  Prag.  ined.  Wchnschr.,  1884.  Langhans,  Cor.-BL  f. 
schweiz.  Aerzte  (1888),  xviii.  Liining  and  Hanau,  ibid.  (1889),  xix. 
Shattock,  Trans.  Path.  Soc.  London,  1885.  Acland,  ibid.,  1886.  Delepine, 
ibid.,  1889.  Harley,  Med.-Chir.  Trans.,  London,  1886.  Crookshank,  ibid., 
1889;  "Manual  of  Bacteriology,"  London,  1896.  Kansome,  Med.-Chir. 
Trans.,  London,  1891.  M'Fadyean,  Journ.  Comp.  Path,  and  Therap., 
1889.  Bostrom,  JSeitr.  z.  path.  Anat.  u.  z.  allg.  Path.,  1890.  Wolff  and 
Israel,  Virchow's  Archiv,  cxxvi.  11.  Illich,  "  Beitrage  zur  Klinik  der 
Aktinomykose,"  Wien,  1892.  Grainger  Stewart  and  Muir,  Edin.  Hosp. 
Rep.,  1893.  Leith,  ibid.,  1894.  Gasperini,  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.  xv.  684.  Hummel,  Beitr.  z.  Tclin.  Chir.  xiii.  No.  3.  Paw- 
lowsky  and  Maksutoff,  Ann.  de  I'lnst.  Pasteur,  vii.  544.  Neukirch, 
Ueber  Strahlenpilze,  Strassburg,  1902.  Doepke,  Munchen.  med.  Wchnschr., 
1902.  Sillurschmidt,  Ztschr.  f.  Hyg.  xxxvii.  345.  J.  Homer  Wright, 
Publications  of  the  Massachusetts  General  Hospital,  Boston,  May  1905. 

Allied  Streptothrices. — Nocard,  Ann.  de  I'lnst.  Pasteur  (1888),  ii.  293. 
Eppinger,  Beitr.  z.  path.  Anat.  u.  z.  allg.  Path.  ix.  287  ;  in  Lubarsch  and 
Ostertag,  "Ergebnisse  der  allgem.  Path."  iii.  328.  Buchholz,  Ztschr.  f. 
Hyg.  xxiv.  470.  Berestnew,  ibid.  xxix.  94.  Cozzolino,  ibid,  xxxiii.  36. 
Flexner,  Journ.  Exper.  Med.  iii.  435.  Dean,  Trans.  Path.  Soc.  London 
(1900),  26.  Birt  and  Leishman,  Journ.  of  Hyg.  ii.  120.  Mertens, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxix.  694.  Foulerton,  Trans. 
Path.  Soc.  London  (1902),  56.  M 'Donald,  Trans.  Med.-Chir.  Soc.  Edin. 
xxiii.  131.  Norris  and  Larkins,  Journ.  Exper,  Med.  v.  155.  Butter- 
field,  Journ.  Infect.  Diseases,  vi.  421. 

MADURA  DISEASE. — Carter  "On  Mycetoma  or  the  Fungus  Disease  of 
India,"  London.  Bassini,  Ref.  in  Centralbl.  f.  Bakteriol.  u.  Parasitenk. 
iv.  652.  Lewis  and  Cunningham,  llth  Ann.  Rep.  San.  Com.  India. 
Kobner,  Fortschr.  d.  Med.  (1886),  No.  17.  Kanthack,  Journ.  Path,  and 
Bacterial,  i.  140.  Boyce  and  Surveyor,  Proc.  Roy.  Soc.  London,  1893. 
Vandyke  Carter,  Trans.  Path.  Soc.  London,  1886.  Vincent,  Ann.  de 
I'lnst.  Pasteur,  viii.  129.  Wright,  J.  H.,  Journ.  Exper.  Med.  iii.  421. 
Oppenheim,  Arch.  f.  Dermat.  u.  Syph.  Ixxi.  209. 


CHAPTER  XIII.— ANTHRAX. 

Bollinger  in  Ziemssen's  "Cyclopaedia  of  Medicine."  Greenfield, 
"Malignant  Pustule"  in  Quain's  "Dictionary  of  Medicine,"  London, 
1894.  Pollender,  Vrtljschr.  f.  gerichtl.  Med.  viii.  ;  Davaine,  Compt. 
rend.  Acad.  d.  sc.  Ivii.  220,  351,  386  ;  lix.  393.  Koch,  Cohu's  Beitr.  z. 
Biol.  d.  Pflanz.  (1876),  ii.  Heft  2.  Mitth.  a.  d.  k.  Gsndhtsamte.  i.  49. 
Pasteur,  Compt.  rend.  Acad.  d.  sc.  xci.  86,  455,  531,  697  ;  xcii.  209. 
Buchner,  Vir  chow's  Archiv,  xci.  Chamberland,  Ann  de  I'lnst.  Pasteur, 
viii.  161.  Chauveau,  Compt.  rend.  Acad.  d.  sc.  xci.  33,  648,  880  ;  xcvi. 
553.  Czaplewski,  Beitr.  z.  path.  Anat.  u.  z.  allg.  Path.  vii.  47. 
Gamaleia,  Ann.  de.  I'lnst.  Pasteur,  ii.  517.  Marshall  Ward,  Proc.  Roy. 
Soc.  London,  Feb.  1893.  Petruschky,  Beitr.  z.  path.  Anat.  u.  z.  allg. 
Path.  iii.  357.  Weyl,  Ztschr.  f.  Hyg.  xi.  381.  Behring,  ibid.  vi.  117  ; 
vii.  171  ;  Osborne,  Arch.  f.  Hyg.  xi.  51.  Roux,  Ann.  de  I'lnst.  Pasteur, 
iv.  25.  Hankin,  Brit.  Med.  Journ.  (1889),  ii.  810 ;  (1890),  ii.  65. 
Hankin  and  Wesbrook,  Ann.  de  I'lnst.  Pasteur,  vi.  633.  Sidney  Martin, 
Rep.  Med.  Off.  Loc.  Govt.  Board  (1890-91),  255.  Marmier,  Ann.  de  I'lnst. 


BIBLIOGRAPHY  581 

Pasteur,  ix.  533.  Rd.  Muir,  Journ.  Path,  and  Bacterial,  v.  374.  Sclavo, 
Rivista  d"  Igiene  e  Sannita  pubblica,  vii.  Nos.  18,  19  ;  Sulla  stato 
presente  della  Sierotherapia  anticarbonchiosa.  Turin,  Pozzo,  1903  (see 
Legge,  Lancet  (1905),  i.  689,  765,  841).  Sobernheim  in  Kolle  and  Wasser- 
mann's  Handbuch,  iv.  793.  Cler,  Gentralbl.  f.  Bakteriol.  und  Para- 
sitenk.  (Orig.)  xl.  241.  Bail,  ibid,  xxxiii.  343,  610.  Sanfelice,  ibid. 
xxxiii.  61.  Roger  and  Gamier,  Compt.  rend.  Soc.  Biol.  Iviii.  863. 
Teacher,  Lancet  (1906),  i.  1306. 


CHAPTER  XIV.— TYPHOID  FEVER,  ETC. 

Eberth,  Virchows  Archiv,  Ixxxi.  58  ;  Ixxxiii.  486.  Koch,  Mitth.  a. 
d.  k.  Gsndhtsamte.  i.  46.  Gaffky,  ibid.  ii.  80.  Klebs,  Arch.  f.  exper. 
Path.  u.  Pharmakol.  xii.  231  ;  xiii.  381.  Escherich,  Fortschr.  d.  Med. 
(1885),  Nos.  16,  17.  Emmerich,  Arch.  f.  Hyg.  iii.  291.  Rodet  and 
Roux,  Arch,  de  med.  exp£r.  et  d'anat.  path.  iv.  317.  Weisser,  Ztschr.  f. 
Hyg.  i.  315.  Klein,  "Micro-organisms  and  Disease,"  London,  1896; 
Rep.  Med.  Off.  LOG.  Govt.  Board  (1892-93),  345  ;  (1893-94),  457  ;  (1894- 
95),  399,  407,  411.  Babes,  Ztschr.  f.  Hyg.  ix.  323.  Vincent,  Compt. 
rend.  Soc.  de  biol.  ser.  ix.  ii.  62.  Birch- Hirschfeld,  Arch.  f.  Hyg.  vii. 
341.  Buchner,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  iv.  353.  Pfuhl, 
ibid.  iv.  769.  Petruschky,  ibid.  vi.  660.  Hunter,  Lancet  (1901),  i.  613. 
Kitasato,  Ztschr.  f.  Hyg.  vii.  515.  Chantemesse  and  Widal,  Bull.  med. 
(1891),  No.  82  ;  Ann.  de  I'lnst.  Pasteur,  vi.  755  ;  vii.  141.  Pere,  Ann. 
de  I'lnst.  Pasteur,  vi.  512.  Neisser,  Ztschr.  /.  klin.  Med.  xxiii.  93. 
Nicholle,  Ann.  de  I'lnst.  Pasteur,  viii.  853.  Quincke  and  Stiihlen, 
Berl.  klin.  Wchnschr.  (1894),  351.  A.  Fraenkel,  Centralbl.  f.  klin.  Med. 
(1886),  No.  10.  E.  Fraenkel  and  Simmonds,  ibid.  (1886),  No.  39. 
Achalme,  Semaine  m<ld.  (1890),  No.  27.  Grawitz,  Charite-Ann.  xvii. 
228.  .Beumer  and  Peiper,  Centralbl.  f.  klin.  Med.  (1887),  No.  4  ;  Ztschr. 
/.  Hyg.  i.  489  ;  ii.  110,  382.  Sirotinin,  ibid.  i.  465.  R.  Pfeitfer  and 
Kolle,  Ztschr.  f.  Hyg.  xxi.  203.  R.  Pfeiffer,  Deutsche  med.  Wchnschr. 
(1894),  898.  Sanarelli,  Ann.  de  I'lnst.  Pasteur,  vi.  721  ;  viii.  193,  353. 
Brieger  and  Fraenkel,  Berl.  klin.  Wchnschr.  (1890),  241,  268.  Brieger, 
Kitasato,  and  Wassermann,  Ztschr.  f.  Hyg.  xii.  137.  Widal,  Semaine 
med.  (1896),  295,  303.  Achard,  ibid.  295,  303.  GrCinbaum,  Lancet, 
Sept.  1896.  Delepine,  Brit.  Med.  Journ.  (1897),  i.  529,  967  ;  Lancet, 
Dec.  1896.  Remlinger  and  Schneider,  Ann.  de  I'lnst.  Pasteur,  xi.  55, 
829.  Widal  and  Sicard,  ibid.  xi.  353.  Peckham,  Journ.  Exper.  Med. 
ii.  549.  Richardson,  ibid.  iii.  329.  Wright  and  Semple,  Brit.  Med. 
Journ.  (1897),  i.  256.  Wright  and  Lamb,  Lancet  (1899),  ii.  1727. 
Wright,  ibid.  (1900),  i.  150;  ii.  1556;  ibid.  (1901),  i.  .609,  858,  1272, 
1532  ;  ii.  715,  1107  ;  ibid.  (1902),  ii.  651  ;  Brit.  Med.  Journ.  (1900),  ii. 
113  ;  ibid.  (1901),  i.  645,  771.  Wright  and  Leishman,  ibid.  (1900),  i. 
622.  See  also  discussion  at  the  Clin.  Soc.  London,  Brit.  Med.  Journ. 
(1901),  ii.  1342.  Sidney  Martin,  ibid.  (1898),  i.  1569,  1644  ;  ii.  11,  73. 
Bokenham,  Trans.  Path.  Soc.  London  (1898),  xlix.  373.  Macfadyen, 
Proc.  Roy.  Soc.  London,  B.  Ixxvii.  548.  Macfadyen  and  Rowland, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  (Orig.)  xxxiv.  618,  765. 
Chantemesse  and  Widal,  Ann.  de  I'lnst.  Pasteur,  vi.  755.  Christophers, 
Brit.  Med.  Journ.  (1898),  i.  71.  Remy,  Ann.  de  I'lnst.  Pasteur,  xiv. 
555,  705.  Wyatt  Johnson,  Brit.  Med.  Journ.  (1897),  i.  231  ;  Lancet 
(1897),  ii.  1746.  Durham,  Lancet  (1898),  i.  154  ;  ibid.  ii.  446. 


582  BIBLIOGRAPHY 

Lorrain  Smith  and  Tennant,  Brit.  Mod.  Journ.  (1899),  i.  193.  Gordon, 
Journ.  Path,  and  Bacterial,  iv.  438.  Castellani,  Ztschrft.  f.  Hyg.  u. 
Infectionskrankh.  xl.  i.  (B.  paratyphosus),  Boycott,  Journ.  Hyg.  vi. 
33.  (Bacillus  Enteritidis,  Gaertner),  refs.  vide  Baumgarten's  Jahres- 
lericht,  iv.  249  ;  vii.  297  ;  xii.  508.  Van  Ermengem,  in  Kolle  and 
Wassermann,  Handbuch,  vol.  ii.  (Psittacosis),  Baumgarten's  Jahres- 
bericht,  xii.  496.  (Bacillus  Enteritidis  Sporogenes),  Klein,  Rep.  Med. 
Off.  Local  Govt.  Board,  xxv.  171  ;  xxvii.  210. 

BACTERIAL  DYSENTERY. — Shiga,  Centralbl.f.  Bakteriol.u.  Parasitenk. 
xxiii.  599  ;  xxiv.  817,  870,  913.  Kruse,  Deutsche  med.  Wchnschr. 
(1900),  637.  Flexner,  Bull.  Johns  Hopkins  Hasp.  (1900),  xi.  39,  231  ; 
Brit.  Med.  Journ.  (1900),  ii.  917.  Strong  and  Musgrave,  Journ.  Amer. 
Med.  Assoc.  (1900),  xxxv.  498.  Vedder  and  Duval,  Journ.  JEJxper.  Med. 
(1902),  vi.  181.  Ogata,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xi.  264. 
See  various  authors  in  Studies  from  the  Rockefeller  Institute  for  Medical 
Research  (1904),  vol.  ii.  Park,  Collins,  and  Goodwin,  Journ.  Med. 
Research  (1904),  xi.  553.  Hiss,  ibid.  (1905),  xiii.  1.  Torrey,  Journ. 
Exper.  Med.  (1905),  vii.  365.  Weaver,  Tunnicliffe,  Heinemann,  and 
Michael,  Journ.  Inf.  Dis.  ii.  70.  Doerr,  Das  Dysenterietoxin, 
Jena,  1907. 

SUMMER  DIARRHEA. — Morgan,  Brit.  Med.  Journ.  (1906),  i.  908; 
(1907),  ii.  16. 

CHAPTER  XV.— DIPHTHERIA. 

Klebs,  Verhandl.  d.  Cong.  f.  innere  Med.  (1883),  ii.  Loffler,  Mitth. 
a.  d.  k.  Gsndhtsamte.  (1884),  421.  Roux  and  Yersin,  Ann.  de  I'lnst. 
Pasteur,  ii.  629  ;  iii.  273  ;  iv.  385.  Brieger  and  Fraenkel,  Berl.  klin. 
Wchnschr.  (1890),  241,  268.  Spronck,  Centralbl.  f.  allg.  Path.  u.  path. 
Anat.  i.  No.  25  ;  iii.  No.  1.  Welch  and  Abbott,  Johns  Hopkins  Hosp. 
Bull.,  1891.  Behring  and  Wernicke,  Ztschr.  f.  Hyg.  xii.  10.  Loffler, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  ii.  105.  v.  Hofmann,  Wien. 
med.  Wchnschr.  (1888),  Nos.  3  and  4.  Cobbett  and  Phillips,  Journ. 
Path,  and  Bacterial,  iv.  193.  Peters,  ibid.  iv.  181.  Wright,  Boston 
Med.  and  S.  Journ.  (1894),  329,  357.  Kanthack  and  Stephens,  Journ. 
Path,  and  Bacteriol.  iv.  45.  Klein,  Brit.  Med.  Journ.  (1894),  ii.  1393  ; 
(1895),  i.  100.  Rep.  Med.  Off.  LOG.  Govt.  Board  (1890-91),  219  ;  (1891- 
92),  125.  Guinochet,  Compt.  rend.  Soc.  de  biol.  (1892),  480.  Roux  and 
Martin,  Ann.  de  I'lnst.  Pasteur,  viii.  609.  Cartwright  Wood,  Lancet 
(1896),  i.  980,  1076  ;  ii.  1145.  Sidney  Martin,  "  Goulstonian  Lectures," 
Brit.  Med.  Journ.  (1892),  i.  641,  696,  755  ;  Rep.  Med.  Off.  LOG.  Govt. 
Board  (1891-92),  147  ;  (1892-93),  427.  Escherich,  Wien.  med.  Wchnschr. 
(1893),  Nos.  47-50  ;  Wien.  klin.  Wchnschr.  (1893),  Nos.  7-10  ;  (1894), 
No.  22  ;  Berl.  klin.  Wchnschr.  (1893),  Nos.  21,  22,  23.  Behring,  "Die 
Geschichte  der  Diphtheric,"  Xeipzig,  1893;  "  Abhandlungen  z.  atiol. 
Therap.  v.  anst.  Krankh.,"  Leipzig,  1893  ;  "  Bekampfung  der  Infections- 
krankheiten,"  Leipzig,  1894.  Ehrlich  and  Wassermann,  Ztschr.  f.  Hyg. 
xviii.  239.  Ehrlich  and  Kossel,  ibid.  xvii.  486.  Ehrlich,  Kossel,  and 
Wassermann,  Deutsche  med.  Wchnschr.  (1894),  353.  Funck,  Ztschr.  f. 
Hyg.  xvii.  401.  Prochaska,  ibid.  xxiv.  373.  Madsen,  ibid.  xxiv.  425. 
Neisser,  ibid.  xxiv.  443.  L.  Martin,  Ann.  de  I'lnst.  Pasteur,  xii.  26. 
Salomonsen  and  Madseu,  ibid.  xii.  763.  Woodhead,  Brit.  Med.  Journ. 
(1898),  ii.  893  ;  Rep.  Metrop.  Asyl.  Bd.,  London,  1901.  Metin,  Ann.  de 
I'lnst.  Pasteur,  xii.  596.  Madsen,  ibid.  xiii.  568,  801.  Dean  and  Todd, 


BIBLIOGRAPHY  583 

Journ.  of  Hyg.  ii.  194.  Cobbett,  ibid.  i.  485.  Graham-Smith,  ibid.  iv. 
258  ;  vi.  286.  Petrie,  ibid.  v.  134.  Rist,  Compt.  rend.  Soc.  de  biol. 
(1903),  No.  25.  Neisser,  Berl.  klin.  Wchnschr.  (1904),  No.  11.  Knapp, 
Journ.  Med.  Research  (1904),  475.  Morgenroth,  Ztschr.  f.  Hyg.  xlviii. 
177.  Bolton,  Lancet  (1905),  i.  1117.  Theobald  Smith,  Journ.  Med. 
Research,  (1905),  xiii.  341.  Boycott,  Journ.  of  Hyg.  v.  223. 


CHAPTER  XVI.— TETANUS,  ETC. 

Nicolaier,  "  Beitrage  zur  Aetiologie  des  Wundstarrkrampfes,"  Inaug. 

Diss.    Gottingen,    1885.     Rosenbach,   Arch.  f.   klin.    Chir.   xxxiv.   306. 

Carle  and  Rattone,  Gior.  d.  r.  Accad.  di  med.  di  Torino,  1884.     Kitasato, 

Ztschr.  f.  Hyg.   vii.  225  ;  x.  267  ;  xii.  256.     Kitasato  and  Weyl,  ibid. 

vi^  41,  404.     Vaillard,  Ann.  de  I'lnst.  Pasteur,  vi.  224,  676.     Vaillard 

and  Rouget,  ibid.  vi.  385.     Behring,  "  Abhandlungen  z.  atiol.  Therap. 

v.  anst.   Krankh.,"  Leipzig,   1893;  Ztschr.  f.  Hyg.  xii.   1,  45;  "Blut- 

serumtherapie,"  Leipzig,  1892;  "  Das  Tetanusheilserum, "  Leipzig,  1892. 

Brieger  and  Fraenkel,  Berl.  klin.  Wchnschr.  (1890),  241,  268.     Sidney 

Martin,  Rep.  Med.  Off.  LOG.   Govt.  Board  (1893-94),  497  ;  (1894-95),  505. 

Uschinsky,   Centralbl.  f.  Bakteriol.   u.   Parasitenk.  xiv.   316.      Tizzoni 

and  Cattani,  Arch.  f.  exper.  Path.  u.  Pharmakol.  xxvii.  432  ;  Centralbl. 

f.  Bakteriol.  u.  Parasitenk.  ix.  189,  685  ;  x.  33,  576  (Ref.)  ;  xi.  325  ; 

Berl.  klin.   Wchnschr.  (1894),  732.     Madsen,  Ztschr.  f.  Hyg.  xxxii.  214. 

Ritchie,    Journ.    of  Hyg.   i.    125.      Danysz,   Ann.    de   VInst.    Pasteur, 

xiii.   155.     Marie  and  Morax,  Ann.  de  I'lnst.  Pasteur,  Paris,  xvi.  818. 

Meyer  and  Ransom,  Proc.  Roy.  Soc.  London,  Ixxii.  26  ;  Arch.  f.  exper. 

Path.   u.  Pharmakl.,  Leipzig,   xlix.    269.      Roux  and  Borrel,  Ann.  de 

I'lnst.  Pasteur,  Paris,  xii.  225  ;  Henderson  Smith,  Journ.  Hyg.  vii.  205. 

Kitt,  see  ref.  in  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  Jena,  Referate, 

xxxii.  359. 

MALIGNANT  (EDEMA. — Pasteur,  Bull.    Acad.   de  med.,   1881,   1887. 

Koch,  Mitth.  a.  d.  k.  Gsndhtsamte.  i.  54.  Kitt,  Jahresb.  d.  k.  Centr.- 
Thierarznei-Schule  in  Miinchen,  1883-84.  W.  R.  Hesse,  Deutsche  med. 
Wchnschr.  (1885),  No.  14.  Chauveau  and  Arloing,  Arch.  v<!t.  (1884), 
366,  817.  Liborius,  Ztschr.  f.  Hyg.  i.  115.  Roux  and  Chamberland, 
Ann.  de  I'lnst.  Pasteur,  i.  562.  Charrin  and  Roger,  Compt.  rend.  Soc. 
de  biol.  (1877),  ser.  viii.  vol.  iv.  p.  408.  Kerry  and  S.  Fraenkel,  Ztschr. 

f.  Hyg.  xii.  204.  Sanfelice,  ibid.  xiv.  339.  Leclainche  and  Velle,  Ann. 
de  VInst.  Pasteur,  xiv.  202,  590. 

BACILLUS  BOTULINUS. — v.  Ermengem,  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.  xix.  443  ;  Ztschr.  f.  Hyg.  xxvi.  1.  Kempner,  ibid.  xxvi. 
481.  Kempner  and  Schepilewsky,  ibid,  xxvii.  213.  Kempner  and 
Pollack,  Deutsche  med.  Wchnschr.  (1897),  No.  32.  Brieger  and  Kempner, 
ibid.  (1897),  No.  33.  Marinesco,  Compt.  rend.  Soc.  de  biol.  (1896),  No.  31. 
Schneidemiihl,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxiv.  577,  619. 
Romer,  ibid,  xxvii.  857. 

QUARTER-EVIL. — See  Nocard  and  Leclainche,  "Les  maladies  micro- 
biennes  des  animaux,"  Paris,  1896.  Arloing,  Cornevin,  et  Thomas, 
"  Le  charbon  symptomatique  du  bceuf,"  Paris,  1887.  Nocard  and  Roux, 
Ann.  de  I'lnst.  Pasteur,  i.  256.  Roux,  ibid.  ii.  49.  See  also  Journ. 
Comp.  Path,  and  Therap.  iii.  253,  346  ;  viii.  166,  233. 

BACILLUS  ^EROGENES  CAPSULATUS. — Welch  and  Nuttall,  Bull.  Johns 
Hopkins  Hosp.  (1892),  81.  Welch  and  Flexner,  Journ.  Exper.  Med.  i.  5. 


584  BIBLIOGRAPHY 

E.  Fraenkel,  Ceniralbl.  f.  Bakteriol.  u.  Parasitenk.  xiii.  13.  Durham, 
Bull.  Johns  Hopkins  Hosp.  (1897),  68.  Norris,  Am.  Journ.  Med.  Sc. 
cxvii.  172. 

CHAPTER  XVII.  —  CHOLERA. 

Koch,  Rep.  of  1st  Cholera  Conference,  1884  (v.  "  Microparasites  in 
Disease,"  New  Sydenham  Soc.,  1886).  Nikati  and  Eietseh,  Compt.  rend. 
Acad.  d.  sc.  xcix.  928,  1145.  Bosk,  Ann.  de  I'Inst  Pasteur,  ix.  507. 
Pettenkofer,  Miinchen.  med.  Wchnschr.  (1892),  No.  46  ;  (1894),  No.  10. 
Sawtsehenko,  Centralbl.  f.  Bakteriol.  u.  ParasitenTc.  xii.  893.  Pfeiffer, 
Ztschr.  f.  Hyg.  xi.  393.  Kolle,  ibid.  xvi.  329.  Issaeff  and  Kolle,  ibid. 
xviii.  17.  Wassermann,  ibid.  xiv.  35.  Sobernheim,  ibid.  xiv.  485. 
Metchnikoff,  Ann.  de  I'Inst.  Pasteur,  vii.  403,  562;  viii.  257,  529. 
Fraenkel  and  Sobernheim,  Hyg.  Rundschau,  iv.  97.  Dunbar,  Arb.  a.  d.  k. 
Gsndhtsamte.  ix.  379.  Pfeiffer  and  Wassermann,  Ztschr.  f.  Hyg.  xiv.  46. 
Wesbrook,  Ann.  de  I'Inst.  Pasteur,  viii.  318.  Scholl,  Berl.  klin.  Wchnschr. 
(1890),  No.  41.  Griiber  and  Wiener,  Arch.f.  Hyg.  xv.  241.  Cunningham, 
Scient.  Mem.  Med.  Off.  India,  1890  and  1894.  Hueppe,  Deutsche  med. 
Wchnschr.  (1889),  No.  33.  Klemperer,  ibid.  (1894),  435 ;  Berl.  klin. 
Wchnschr.  (1892),  969.  Lazarus,  ibid.  (1892),  1071.  Reincke,  Deutsche 
med.  Wchnschr.  (1894),  795.  Koch,  Ztschr,  f.  Hyg.  xiv.  319.  Voges, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xv.  453.  Pastana  and  Bettencourt, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xvi.  401.  Dieudonne,  ibid.  xiv. 
323.  Celliand  Santori,  ibid.  xv.  289.  Neisser,  ibid.  xiv.  666.  Sanarelli, 
Ann.  de  I'Inst.  Pasteur,  vii.  693.  Ivanoff,  Ztschr.  f.  Hyg.  xv.  485. 
Issaeff,  ibid.  xvi.  286.  Pfuhl,  ibid.  x.  510.  Rumpel,  Deutsche  med. 
Wchnschr.  (1893),  160.  Klein,  Rep.  Med.  Off.  LOG.  Govt.  Board,  1893  ; 
"Micro-organisms  and  Disease,"  London,  1896.  Haffkine,  Brit.  Med. 
Journ.  (1895),  ii.  1541  ;  Indian  Med.  Gaz.  (1895),  No.  1  ;  "Anti-cholera 
Inoculation,"  Rep.  San.  Com.  India,  Calcutta,  1895.  Pfeiffer  in 
Fliigge,  "Die  Mikroorganismen,"  3rd  ed.  1896;  Gamaleia,  Ann.  de 
I'Inst.  Pasteur,  ii.  482,  552.  Achard  and  Bensande,  Semaine  mtd. 
(1897),  151.  Rumpf,  "Die  Cholera  Asiatica  und  Nostras,"  Jena,  1898. 
Kraus  and  Pribram,  Centralbl.  f.  Bakteriol.  xli.  (Orig.),  15,  155.  Kraus 
and  Prantschoff,  ibid.  377,  480.  A.  Macfadyen,  ibid.  xlii.  (Orig.), 
365.  Gotschlich,  Scientific  Reps.  Sanitary,  Maritime,  and  Quarantine 
Council  of  Egypt,  Alexandria,  1905,  1906.  For  discussion,  vide  Supple- 
ment to  Centralbl.  f.  'Bakteriol.  Referate,  xxxviii.  84.  Dunbar,  Berlin, 
klin.  Wchnschr.  (1902),  No.  39. 


CHAPTER  XVIII.— INFLUENZA,  ETC. 

INFLUENZA. — Pfeiffer,  Kitasato,  and  Canon,  Deutsche  med.  Wchnschr. 
xviii.  28,  and  Brit.  Med.  Journ.  (1892),  i.  128.  Babes,  Deutsche  med. 
Wchnschr.  xviii.  113.  Pfeiffer  and  Beck,  ibid.  (1892),  465.  Pfuhl, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xi.  397.  Klein,  Rep.  Med.  Off. 
Loc.  Govt.  Board  (1893),  85.  Pfeiffer,  Ztschr.  f.  Hyg.  xiii.  357.  Huber, 
Ztschr.  f.  Hyg.  xv.  454.  Kruse,  Deutsche  med.  Wchnschr.  (1894),  513. 
Pelicke,  Berl.  klin.  Wchnschr.  (1894),  524.  Pfuhl  and  Walter,  Deutsche 
med.  Wchnschr.  (1896),  82,  105.  Cantani,  Ztschr.  f.  Hyg.  xxiii.  265. 
Pfuhl,  Ztschr.  f.  Hyg.  xxvi.  112.  Wassermann,  Deutsche  med.  Wchnschr. 
(1900),  No.  28.  Clemens,  Miinchen.  med.  Wchnschr.  (1900),  No.  27. 


BIBLIOGRAPHY  585 

Wynecoop,  Journ.  filed.  Ass.,  February  1903.  Neisser,  Deutsche  med. 
Wchnschr.  (1903),  No.  26.  Auerbach,  Ztschr.  f.  Hyg.  (1904),  xlviii.  259. 

WHOOPING-COUGH. — Jochmann,  Arch.  f.  klin.  Med.  Ixxxiv.  470. 
Spengler,  Deutsche  med.  Wchnschr.  (1897),  830.  Davis,  Journ.  Infect. 
Dis.  iii.  1.  Bordet  and  Gengou,  Ann.  de  I'Inst.  Pasteur,  xx.  731. 

PLAGUE. — Kitasato,  Lancet  (1894),  ii.  428.  Yersin,  Ann.  de  I'Inst. 
Pasteur,  viii.  662.  Lowson,  Lancet  (1895),  ii.  199.  Yersin,  Calmette, 
and  Borrel,  Ann.  de  I'Inst.  Pasteur,  ix.  589.  Aoyama,  Centralbl.  f. 
Bakteriol.  u.  ParasitenJc.  xix.  481.  Zettnow,  Ztschr.  f.  Hyg.  xxi.  164. 
Yersin,  Ann.  de  I'Inst.  Pasteur,  xi.  81.  Gordon,  Lancet  (1899),  i.  688. 
Simond,  Ann.  de  I'Inst.  Pasteur,  xii.  625.  Haff kine,  Brit.  Med.  Journ. 
(1897),  i.  424.  Wyssokowitz  and  Zabolotny,  Ann.  de  I'Inst.  Pasteur, 
xi.  663.  Ogata,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxi.  769.  Childe, 
Brit.  Med.  Journ.  (1898),  ii.  858.  See  also  Brit.  Med.  Journ.  and 
Lancet,  1897-99.  Lustig  and  Galeotti,  Deutsche  med.  Wchnschr.  (1897), 
No.  15.  Markl,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxiv.  641,  728  ; 
xxix.  810.  Cairns,  Lancet  (1901),  i.  1746.  Montenegro,  "  Bubonic 
Plague,"  London,  1900.  Netter,  "La  peste  et  son  bacille,"  Paris,  1900. 
Mitth.  der  Deutschen  Pest-Kommission,  Deutsche  med.  Wchnschr.  (1897), 
Nos.  17,  19,  31,  32.  "Report  of  the  Indian  Plague  Commission  (1898- 
99),"  London,  1900-1901.  Also  numerous  papers  in  the  Lancet  and  Brit. 
Med.  Journ.,  1897-1901.  Regarding  Glasgow  epidemic  see  ibid.  (1900), 
ii.  "Reports  on  Plague  Investigations  in  India,"  Journ.  Hyg.  (1906), 
vi.  422  ;  (1907),  vii.  323. 

RELAPSING  FEVER. — Obermeier,  Centralbl.  f.  d.  med.  Wissensch. 
(1873),  145  ;  and  Berl.  klin.  Wchnschr.  (1873),  No.  35.  Miinch, 
Centralbl.  f.  d.  med.  Wissensch.,  1876.  Koch,  Deutsche  med.  Wchnschr. 
(1879),  327.  Moczutkowsky,  Deutsches  Arch.  f.  klin.  Med.  xxiv.  192. 
Vandyke  Carter,  Med.-Chir.  Trans.,  London  (1880),  78.  Lubinoff, 
Virclwws  Archiv,  xcviii.  160.  Metchnikotf,  ibid.  cix.  176.  Soudake- 
witch,  Ann.  de  I'Inst.  Pasteur,  v.  545.  Lamb,  Scient.  Mem.  Med.  Off. 
India  (1901),  pt.  xii.  77.  Sawtschenko  and  Melkich,  Ann.  de  I'Inst. 
Pasteur,  xv.  497.  Tictin,  Centralbl.  f.  Bakteriol.  xxi.  179.  Karlinski, 
Centralbl.  f.  Bakteriol.  (1902),  Orig.  xxxi.  566.  Gabritschewsky,  Ztschr. 
f.  klin.  Med.  (1905),  Bd.  56.  Norris,  Pappenheimer,  Flournoy,  Journ. 
Infect.  Dis.  iii.  266.  Novy  and  Knapp,  ibid.  291.  Zettnow,  Ztschr.  f. 
Hyg.  (1906),  Hi.  485  ;  Deutsche  med.  Wchnschr.,  1906. 

AFRICAN  TICK  FEVER.— Ross  and  Milne,  Brit.  Med.  Journ.  (1904), 
ii.  1453.  Button  and  Todd,  Thompson-  Yates  Laboratory  Rep.  (1905),  vi. 
pt.  ii.  Koch,  Deutsche  med.  Wchnschr.,  1905  ;  Berl.  klin.  Wchnschr.,  1906. 
Hodges  and  Ross,  Brit.  Med.  Journ.  (1905),  i.  713.  Breuil  and  Kinghorn, 
ibid.  i.  668.  Breuil,  Lancet  (1906),  i.  1806.  Levaditi,  Compt.  rend.  Soc. 
biol.,  1906. 

MALTA  FEVER. — Bruce,  Practitioner,  xxxix.  160  ;  xl.  241  ;  Ann.  de 
I'Inst.  Pasteur,  vii.  291.  Bruce,  Hughes,  and  Westcott,  Brit.  Med. 
Journ.  (1887),  ii.  58.  Hughes,  Ann.  de  I'Inst.  Pasteur,  vii.  628  ;  Lancet 
(1892),  ii.  1265.  Wright  and  Semple,  Brit.  Med.  Journ.  (1897),  i.  1214. 
Wright  and  Smith,  ibid.  (1897),  i.  911  ;  Lancet  (1897),  i.  656.  Welch, 
ibid.  (1897),  i.  1512.  Gordon,  ibid.  (1899),  i.  688.  Durham,  Journ. 
Path,  and  Bacteriol.  v.  377.  Bruce  in  Davidson's  "  Hygiene  and 
Diseases  of  Warm  Climates,"  Edinburgh  and  London,  1893.  Birt  and 
Lamb,  Lancet  (1899),  ii.  701.  Brunner,  Wien.  klin.  Wchnschr.  (1900), 
No.  7.  Bruce,  Journ.  Roy.  Army  Med.  Corps  (1904),  ii.  487,  731  ;  (1907), 
viii.  225.  Horrocks,  Proc.  Roy.  Soc.  London,  Series  B  (1905),  Ixxvi. 


586  BIBLIOGRAPHY 

510.  "Reports  of  the  Commission  on  Mediterranean  Fever,"  1904-1907 
(reprinted  in  Journ.  Roy.  Army  Med.  Corps).  Eyre  in  Kolle  and 
Wassermann's  Handbuch  d.  Pathog.  Mikro-organismen,  Erganzungsband, 
1906. 

YELLOW  FEVER. — Sternberg,  Rep.  Am.  Pub.  Health  Ass.  xv.  170. 
Sanarelli,  Ann.  de  I'Inst.  Pasteur,  xi.  433,  673,  753  ;  xii.  348.  David- 
son, art.  in  Clifford  Allbutt's  "System  of  Medicine,"  vol.  ii.,  London, 
]897.  Sternberg,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxii.  145  ;  xxiii. 
769.  Sanarelli,  ibid.  xxii.  668.  Keed  and  Carroll,  Medical  News,  April 
1899.  Reed,  Journ.  of  Hyg.  ii.  101  (with  full  references).  Durham, 
Thompson-  Yates  Laboratory  Rep.  (1902),  iv.  pt.  ii.  485.  Gorgas,  Lancet, 
1902,  Sept.  9  ;  1903,  March  28.  Marchoux,  Salimbeni,  and  Simond, 
Ann.  de  I'Inst.  Pasteur,  xvii.  665  ;  xx.  16,  104,  161.  Bandi,  Ztschr.  f. 
Hyg.  (1904),  xlvi.  81.  Otto  and  Neumann,  Ztschr.  f.  Hyg.  (1905),  Ii. 
Heft  3.  Reed,  Carroll,  Agramonte,  Lazear,  Proc.  Amer.  Health  Ass., 
1900;  Journ.  Amer.  Med.  Ass.,  Feb.  1901.  Carroll,  New  York  Med. 
Journ.,  Feb.  1904  ;  Amer.  Medicine  (1906),  xi.  383. 


CHAPTER  XIX.— IMMUNITY. 

For  early  inoculation  methods  (e.g.  against  anthrax,  chicken  cholera, 
etc.),  see  " Microparasites  in  Disease,"  New  Syd.  Soc.  1886.  Duguid 
and  Sanderson,  Journ.  Roy.  Agric.  Soc.  (1880),  267.  Greenfield,  ibid. 
(1880),  273  ;  Proc.  Roy.  Soc.  London,  June  1880.  Toussaint,  Compt. 
rend.  Acad.  d.  sc.  xci.  135.  Haffkine,  Brit.  Med.  Journ.  (1891),  ii. 
1278.  Klein,  ibid.  (1893),  i.  632,  639,  651.  Klemperer,  Arch.  f.  exper. 
Path.  u.  Pharmakol.  xxxi.  356.  Buchner,  Milnchen.  med.  Wchnschr. 
(1893),  449,  480.  Ehrlich,  Deutsche  med.  Wchnschr.  (1891),  976,  1218. 
R.  Pfeiffer,  Ztschr.  /.  Hyg.  xviii.  1  ;  xx.  198.  Pfeiffer  and  Kolle,  ibid. 
xxi.  203.  Bordet,  Ann.  de  I'Inst.  Pasteur,  ix.  462  ;  xi.  106.  Metehni- 
koff,  Firchow's  Archiv,  xcvi.  177  ;  xcvii.  502  ;  cvii.  209 ;  cix.  17,6  ; 
Ann.  de  I'Inst.  Pasteur,  iii.  289  ;  iv.  65  ;  iv.  193  ;  iv.  493  ;  v.  465  ;  vi. 
289  ;  vii.  402  ;  vii.  562  ;  viii.  257  ;  viii.  529  ;  ix.  433.  Calmette,  Ann. 
de  I'Inst.  Pasteur,  viii.  275  ;  xi.  95.  Fraser,  Proc.  Roy.  Soc.  Edin.  xx. 
448.  Marmorek,  Ann.  de  I'Inst.  Pasteur,  ix.  593.  Metchnikoff,  Roux, 
and  Taurelli-Salimbeni,  ibid.  x.  257.  Charrin  and  Roger,  Compt.  rend. 
Soc.  de  biol.  (1887),  667.  Griiber  and  Durham,  Milnchen.  med.  Wchnschr. 
(1896),  March.  Durham,  Journ.  Path,  and  Bacteriol.  iv.  13.  Cart- 
wright  Wood,  Lancet  (1896),  i.  980  ;  ii.  1145.  Sidney  Martin,  "Serum 
Treatment  of  Diphtheria,"  Lancet  (1896),  ii.  1059.  Ransome,  "On 
Immunity  to  Disease,"  London,  1896.  Burdon  Sanderson,  "  Croonian 
Lectures,"  Brit.  Med.  Journ.  (1891),  ii.  983,  1033,  1083,  1135.  Discus- 
sion on  Immunity,  Path.  Soc.  London,  Brit.  Med.  Journ.  (1892),  i.  373. 
Fodor,  Deutsche  med.  Wchnschr.  (1887),  No.  34.  Hueppe,  Berl.  klin. 
Wchnschr.  (1892),  No.  17.  Nicholle,  Ann.  de  I'Inst.  Pasteur,  xii.  161. 
Salomonsen  and  Madsen,  ibid.  xi.  315  ;  xii.  763.  Roux  and  Borrell,  ibid. 
xii.  225.  Salimbeni,  ibid.  xi.  277.  Wassermann  and  Takaki,  Berl. 
klin.  Wchnschr.  (1898),  xxxv.  4.  Blumenthal,  Deutsche  med.  Wchnschr. 
xxiv.  185.  Ransom,  ibid.  xxiv.  117.  Meade  Bolton,  Journ.  Exper. 
Med.  i.  543.  Fraser,  T.  R.,  Brit.  Med.  Journ.  (1895),  i.  1309  ;  ii.  415, 
416  ;  (1896),  i.  957  ;  (1896),  ii.  910  ;  (1897),  ii.  125,  595.  Calmette, 
Ann.  de  I'Inst.  Pasteur,  vi.  160,  604  ;  viii.  275  ;  ix.  225  ;  x.  675  ;  xi. 
214  ;  xii.  343.  C.  J.  Martin,  Journ.  Physiol.  xx.  364  ;  Proc.  Roy.  Soc. 


BIBLIOGRAPHY  587 

London,  Ixiv.  88.  C.  J.  Martin  and  Cherry,  ibid.  Ixiii.  420.  Gautier, 
"Les  Toxines  microbiennes  et  animales,"  Paris,  1896.  Wassermann, 
Berl.  klin.  Wchnschr.  (1898),  1209.  Pfeiffer  and  Marx,  Ztschr.  f.  Hyg. 
xxvii.  272.  Bordet,  Ann.  de  VInst.  Pasteur,  xii.  688.  Ehrlich,  Deutsche 
med.  Wchnschr.  (1898),  xxiv.  597.  "Die  Wertbemessung  des  Diph- 
therieheilserums,"  Jena,  1897  ;  Croonian  Lecture,  Proc.  Roy.  Soc. 
London,  Ixvi.  424  ;  Deutsche  med.  Wchnschr.  xxvii.  (1901),  866,  888, 
913;  Nothnagel's  "  Specielle  Pathologic  und  Therapie,"  Bd.  viii. 
Schlussbetrachtungen.  Ehrlich  and  Morgenroth,  Berl.  klin.  Wchnschr. 
(1899),  xxxvi.  6,  481  ;  (1900),  xxxvii.  453,  681  ;  (1901),  xxxviii.  251, 
569,  598.  Weigert,  in  Lubarsch  and  Ostertag,  "  Ergebnisse  der 
Allgemeinen  Pathologic  "  (1897),  iv.  Jahrg.  (Wiesbaden,  1899).  Morgen- 
roth, Oentralbl.  f.  Bakteriol.  u.  Parasitenk.  xxvi.  349.  Bulloch,  Trans. 
Jenner  Inst.  2nd  ser.  p.  46.  Donitz,  Deutsche  med.  Wchnschr.  (1897), 
xxiii.  428.  Bordet,  Ann.  de  rinst.  Pasteur,  xii.  688  ;  xiii.  225,  273  ; 
xiv.  257  ;  xv.  303  ;  xvii.  161  ;  xviii.  593  ;  Metchnikoff,  ibid.  xiii.  737  ; 
xiv.  369  ;  xv.  865.  Gengou,  ibid.  xv.  232.  Sawtschenko,  ibid.  xvi.  106. 
Ritchie,  Journ.  of  Hyg.  ii.  215,  251,  452  (with  full  references)  ; 
"General  Pathology  of  Infection,"  in  Clifford  Allbutt's  "System  of 
Medicine,"  2nd  ed.  1906,  vol.  ii.  pt.  i.  p.  1.  Metchnikoff,  "  L'inmiunite 
dans  les  maladies  infectieuses,"  Paris,  1901.  Neisser  and  Wechsberg, 
Munchen.  med.  Wchnschr.  1901,  No.  18.  Von  Dungern,  ibid.  (1899), 
1288  ;  (1900),  677,  973.  Ehrlich,  Collected  Studies  on  Immunity 
(English  trans.),  1906,  Bordet.  Joos,  Ztschr.  f.  Hyg.  xxxvi.  422;  xl. 
203  ;  Centralbl.  f.  Bakteriol.  (Orig.)  xxxiii.  762.  Eisenberg  and  Volk, 
Ztschr.  f.  Hyg.  xl.  155.  Dreyer  and  Jex-Blake,  Journ.  Path,  and  Bacterial. 
xi.  1.  (Precipitins)  Nuttall,  Blood  Immunity  and  Blood  Relationship, 
Cambridge,  1904. 

OPSONINS.— Denys  and  Leclef,  La  cellule,  1895,  177.  Sawtschenko, 
Ann.  de  rinst.  Pasteur,  1902,  106.  Wright  and  Douglas,  Proc.  Roy. 
Soc.  London,  Ixxii.  357  ;  Ixxiii.  128  ;  Ixxiv.  147.  Wright  and  Reid,  ibid. 
Ixxvii.  211.  Bulloch  and  Atkiv,ibid.  Ixxiv.  379.  Bulloch  and  Western, 
ibid.  Ixxvii.  531.  Neufeld  and  Rimpau,  Deutsche  med.  Wchnschr.  (1904), 
1458.  Hektoen  and  Ruediger,  Journ.  Infect.  Diseases,  1905,  128. 
Leishman,  Trans.  Path.  Soc.  Lond.  1905.  Muir  and  Martin,  Brit.  Med. 
Journ.  1906,  ii.  ;  Proc.  Roy.  Soc.  London,  Ixxix.  187. 


APPENDIX  A.— SMALLPOX. 

Jenner,  "An  Inquiry  into  the  Causes  and  Effects  of  the  Variola 
Vaccine,"  London,  1798.  Creighton,  art.  "Vaccination"  inEncy.  Brit., 
9th  ed.  Crookshank,  "  Bacteriology  and  Infective  Diseases."  M'Vail, 
"Vaccination  Vindicated."  Chauveau,  Viennois  et  Mairet,  "Vaccine 
et  variole,  nouvelle  etude  experimentale  sur  la  question  de  1'identite  de 
ces  deux  affections,"  Paris,  1865.  Klein,  Rep. Med.  Off.  Loc.  Govt.  Board 
(1892-93),  391  ;  (1893-94),  493.  Copeman,  Brit.  Med.  Journ.  (1894),  ii. 
631  ;  Journ.  Path,  and  Bacteriol.  ii.  407  ;  art.  in  Clifford  Allbutt's 
"System  of  Medicine,"  vol.  ii.  L.  Pfeiffer,  "Die  Protozoen  als  Krank- 
heitserreger,"  Jena,  1891.  Ruffer,  Brit.  Med.  Journ.  (1894),  June  30. 
Beclere,  Chambon,  and  Menard,  Ann.  de  I' Inst.  Pasteur,  x.'l  ;  xii.  837. 
Copeman,  "Vaccination,"  London,  1899.  Calmette  and-Guerin,  Ann. 
de  rinst.  Pasteur,  xv.  161.  Guarnieri,  Centralbl.  f.  Bakteriol.  u.  Para- 
sitenk. xvi.  299.  Ewing,  Journ.  Med.  Research,  xiii.  233.  Prowazek, 


588  BIBLIOGRAPHY 

Arb.  a.  d.  kaiserl.  Gesundheilsamte,  xxii.  535  ;  xxiii.  525.  Wasielewski, 
Ztschrft.  f.  Hyg.  xxxviii.  212.  Bonhoff,  Berl.  klin.  Wchnschrft.  1905, 
p.  1142.  Carini,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  (Orig.)  xxxix.  685. 


APPENDIX  B. — HYDROPHOBIA. 

Pasteur,  Compt.  rend.  Acad.  d.  sc.  xcii.  1259  ;  xcv.  1187  ;  xcviii.  457. 
1229  ;  ci.  765  ;  cii.  459,  835  ;  ciii.  777.  Schaffer,  Ann.  de  I'lnst.  Pasteur, 
iii.  644.  Fleming,  Trans.  7th  Internal.  Cong.  Hyg.  and  Deinog.  iii.  16. 
Helman,  Ann.  de  I'lnst.  Pasteur,  ii.  274  ;  iii.  15.  Babes  and  Lepp,  ibid. 
iii.  384.  Nocard  and  Roux,  ibid.  ii.  341.  Roux,  ibid.  i.  87  ;  ii.  479. 
Bruschettini,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xx.  214  ;  xxi.  203. 
Memmo,  ibid.  xx.  209  ;  xxi.  657.  Frantzius,  ibid,  xxiii.  782  ;  xxiv. 
971.  Remlinger,  Ann.  de  I'lnst.  Pasteur,  xvii.  834  ;  xviii.  150  ;  xix. 
625.  Negri,  Ztschrft.  f.  Hyg.  u.  Infectionskrankh.  xliii.  507  ;  xliv.  519. 
Williams  and  Lowden,  Journ.  Inf.  Dis.  iii.  452. 


APPENDIX  C.— MALARIAL  FEVER. 

Laveran,  Bull.  Acad.  de  med.  (1880)  ser..ii.  vol.  ix.  1346  ;  "Traitedes 
fievres  palustres,"  Paris,  1884  ;  "  Du  paludisme  et  de  son  hernatozoaire," 
Paris,  1891.  Marchiafava  and  Celli,  Fortschr.  d.  Med.,  1883  and  1885  ; 
also  in  Virchows  Festschrift.  Golgi,  Arch,  per  le  sc.  med.,  1886  and  1889  ; 
Fortschr.  d.  Med.  (1889),  No.  3  ;  Ztschr.  f.  Hyg.  x.  136  ;  Deutsche  med. 
Wchnschr.  (1892),  663,  685,  707,  729  ;  Steinberg,  New  York  Med.  Rec. 
xxix.  No.  18.  James,  ibid,  xxxiii.  No  10.  Councilman,  Fortschr.  d.  Med. 
(1888),  Nos.  12,  13.  Osier,  Trans.  Path.  Soc.  Philadelphia,  xii.  xiii. 
Grass!  and  Feletti,  Riforma  med.  (1890),  ii.  No.  50.  Caualis,  Fortschr.  d. 
Med.  (1890),  Nos.  8,  9.  Danilewsky,  Ann.  de  I'lnst.  Pasteur,  xi.  758. 
"  Parasites  of  Malarial  Fevers,"  New.  Syd.  Soc.,  1894  (Monographs  by 
Marchiafava  and  Bignami,  and  by  Mannaberg,  with  Bibliography). 
Manson,  Brit.  Med.  Journ.  (1894)  i.  1252,  1307  ;  Lancet  (1895),  ii.  302  ; 
Brit.  Med.  Journ.  (1898),  ii.  849  ;  Koch,  Berl.  klin.  Wchnschr.  (1899), 
69.  Ross,  Indian  Med.  Gaz.  xxxiii.  14,  133,  401,  448.  Nuttall, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxv.  877,  903  ;  xxvi.  140  ;  xxvii. 
193,  218,  260,  328  (with  full  literature).  Manson,  Lancet  (1900), 
i.  1417;  (1900)  ii.  151.  Gray,  Brit.  Med.  Journ.  (1902),  i.  1121. 
Leishman,  ibid.  (1901),  i.  635  ;  ii.  757.  Daniel,  ibid.  (1901),  i.  193. 
Celli,  ibid.  (1901),  i.  1030.  Nuttall  and  Shipley,  Journ.  of  Hyg.  i.  45, 
269,  451  (with  literature).  Ross,  Nature,  Ixi.  522  ;  "  Mosquito  Brigades 
and  how  to  organise  them,"  London,  1902.  Celli,  "Malaria,"  trans,  by 
Eyre,  London,  1900.  Lankester,  Brit.  Med.  Journ.  (1902),  i.  652. 
Ewing,  Journ.  Exper.  Med.  v.  429  ;  vi.  119.  Schaudinn,  Arbeit,  ans  d. 
kaiserl.  Gesundheitsamte,  xix.  ;  Argutinsky  Archiv  mikroskop.  Anat. 
lix.  315  ;  Ixi.  331.  Ruge  in  Kolle  and  Wassermann's  Handbuch  d. 
pathogen.  Mikroorganismen,  Erganzungsband,  1907  (full  literature). 
Ross,  Lancet  (1903),  i.  86.  Minchin,  "The  Sporozoa,"  London,  1903. 
Stephens,  art.  "  Blackwater  Fever,"  in  Allbutt's  "System  of  Medicine," 
vol.  ii.  pt.  ii.,  London,  1907. 


BIBLIOGRAPHY  589 


APPENDIX  D. — DYSENTERY. 

Losch,  Virchow's  Archiv,  Ixv.  196.  Cunningham,  Quart.  Journ. 
Micr.  Sc.,  N.S.  xxi.  234.  Kartulis,  Virchow's  Archiv,  cv.  118  ;  CentralbL 
f.  Baktcriol.  u.  Parasitenk.  ii.  745  ;  ix.  365.  Koch,  Arb.  a.  d.  k. 
Gsndhtsamte.  iii.  65.  Councilman  and  Lafleur,  Johns  Hopkins  Hosp. 
Rep.  (1891),  ii.  395.  Maggiora,  CentralbL  /.  Bakteriol.  u.  Parasitenk. 
xi.  173.  Ogata,  ibid.  xi.  264.  Schuberg,  ibid.  xiii.  598,  701.  Quincke 
and  Roos,  Berl.  klin.  Wchnschr.  (1893),  1089.  Kruse  and  Pasquale, 
Ztschr.  /.  Hyg.  xvi.  i.  Ciechanowski  and  Nowak,  CentralbL  f.  Bakteriol. 
u.  Parasitenk.  xxiii.  445.  Howard  and  Hoover,  Am.  Journ.  Med.  Sc. 
(1897),  cxiv.  150,  263.  Harris,  Fir  chow's  Archiv,  clxvi.  67.  Schaudinn, 
Arbeit,  ans  d.  kaiserl.  Gsndhtsamte.  (1903),  xix.  547.  Lesage,  Ann. 
de  rinst.  Pasteur  (1905),  xix.  9.  Kartulis  in  Kolle  and  Wassermann' s 
Handbuch  d.  pathog.  Mikroorganismen,  Erganzungsband,  1906  ;  Cen- 
tralbL f.  Bakteriol.  (Originate)  1904,  xxxvii.  527. 


APPENDIX  E. — TEYPANOSOMIASIS,  ETC. 

GENERAL. — Laveran  and  Mesnil,  Try pano somes  et  trypanosomiasis, 
Paris,  Masson,  1904.  Minchin,  in  Clifford  Allbutt's  "System  of  Medicine," 
2nd  ed.  vol.  ii.  pt.  ii.  p.  9,  London,  Macmillan,  1907.  Schaudinn, 
Arbeit,  a.  d.  kaiserl.  Gesundheitsamte,  xx.  387.  Mense,  Handbuch  der 
Tropenkrankheiten,  Leipzig,  1906,  Barth.  Novy  and  MacNeal,  Journ. 
Inf.  Dis.  ii.  256.  Leishman,  Journ.  Hyg.  iv.  434. 

SLEEPING  SICKNESS. — Mott,  Reports  of  the  Sleeping  Sickness  Com- 
mission of  the  Royal  Society,  pt.  vii.  No.  15,  London,  Bale,  Sons  and 
Danielsson,  1906.  Button  and  Todd,  Brit.  Med.  Journ.  (1903),  i.  304. 
Dutton  and  Todd,  Thompson-  Yates  Lab.  Rep.  v.  pt.  ii.  i.  ;  v.  pt.  ii.  97. 
Dutton,  Todd,  and  Christy,  ibid.  vi.  pt.  i.  p.  1.  Manson  and  Daniels,  ibid. 
(1903),  i.  1249.  Idem,  ibid.  (1903),  ii.  1461.  Low  and  Mott,  ibid.  (1904),  i. 
1000.  Bettencourt,  Kopke,  Resende,  and  Mendes,  ibid.  (1903),  i.  908. 
Castellani,  Reports  of  the  Sleeping  Sickness  Commission  of  the  Royal 
Society,  No.  1,  i.  1,  London,  Harrison  and  Sons,  1903.  Bruce  and 
Nabarro,  ibid.  (1903),  No.  1,  ii.  11.  Bruce,  Nabarro,  and  Greig 
ibid.  (1903),  No.  4,  viii.  3.  Greig  and  Gray,  ibid.  (1905),  No.  6,  ii. 
3.  Leishman,  Journ.  Hyg.  iv.  434.  Minchin,  Gray,  and  Tulloch, 
Reports  of  the  Sleeping  Sickness  Commission  of  the  Royal  Society,  No. 
8,  xxi.  122,  London,  H.M.  Stationery  Office,  1907.  Manson,  Brit. 
Med.  Journ.  (1903),  ii.  1249,  1461.  See  Discussions  at  British  Medical 
Association,  Brit.  Med.  Journ.  (1903),  ii.  637  ;  (1904),  ii.  365.  Thomas, 
Thompson- Yates  Lab.  Rep.  vi.  pt.  ii.  1. 

KALA-AZAR. — Leishman, .  Brit.  Med.  Journ.  (1903),  i.  1252.  Idem, 
Clifford  Allbutt,  "System  of  Medicine,"  2nd  ed.  vol.  ii.  pt.  ii.  226,  London, 
Macmillan,  1907.  Idem,  Mense,  Handbuch  der  Tropenkrankheiten,  iii. 
591,  Leipzig,  Barth.,  1906.  Leishman  and  Statham,  Journ.  of  Roy. 
Army  Med.  Corps,  iv.  321.  Donovan,  Brit.  Med.  Journ.  (1903),  ii.  79. 
Rogers,  Quart.  Journ.  Micr.  Soc.  xlviii.  367.  Idem,  Brit.  Med.  Journ. 
(1904),  i.  1249  ;  ii.  645.  Idem,  Proc.  Roy.  Soc.  Ixxvii.  284.  Bent^,y 


590  BIBLIOGRAPHY 

Brit.  Med.  Journ.  (1904),  ii.  653  ;  ibid.  (1905),  i.  705.  Christophers, 
Scientif.  Mem.  by  Off.  of  the  Med.  and  San.  Dept.  of  the  Govt.  of  India, 
Nos.  8,  11,  15.  Ross,  Brit.  Med.  Journ.  (1903),  ii.  1401.  See  discussion 
at  Brit.  Med.  Assoc.,  Brit.  Med.  Journ.  (1904),  ii.  642. 

DELHI  SORE. — Wright,  J.  H.,  Journ.  Med.  Research,  x.  472. 

PIROPLASMOSIS. — See  Minchin,  loc.  cit.  supra.  Koch,  Deutsche,  med. 
Wchnschrft.  (1905),  No.  47  ;  Ztschrft.  f.  Hyg.  u.  InfektionsTcranTch.  liv.  i. 
Nuttall,  Journ.  Hyg.  iv.  219.  Kuttall  and  Graham-Smith,  ibid.  v. 
237  ;  vi.  586. 


INDEX 


INDEX 


Abrin,  169 

immunity  against,  464,  469 
Abscesses  (v.  also  Suppuration) 

bacteria  in,  174 

in  dysentery,  540 
Absolute  alcohol,  fixing  by,  89 
Acid-fast  bacilli,  239,  252 

stain  for,  100 

Acid  formation,  observation  of,  44,  77 
Acquired  immunity  in  man,  456 

theories  of,  490 
Actinomyces,  15 

characters  of,  287 

cultivation  of,  292 

inoculation  with,  296 

varieties  of,  294 
Actinoniycosis,  286 

anaerobic  streptothrices  in,  293 

diagnosis  of,  296 

lesions  in,  290 

origin  of,  292 
Active  immunity,  458,  459 
Aerobes,  17 

culture  of,  57 

separation  of,  51. 
JUstivo-autumnal  fevers,  529 
Agar  media  (v.  also  Culture  media),  35 

separation  by,  55 
Agglutinable  substance,  486 
Agglutination  by  sera,  485 

in  relapsing  fever,  442 

methods,  109 

of  b.  mallei,  282 

of  b.  typhosus,  etc.,  337 

of  cholera  vibrio,  412 

of  m.  melitensis,  450 

of  plague  bacillus,  437 

of  red  blood  corpuscles,  481,  485 

theories  regarding,  485 

38  593 


Agglutinins,    primary    (homologous), 

342 

secondary  (heterologous),  342 
Agglutinogen,  486 
Agglutinoids,  486 
Aggressins,  164 
Air,  bacteria  in,  126 

examination  of,  for  bacteria,  126 
Albumose  of  anthrax,  immunity  by, 

312 
Albumoses,  165 

in  diphtheria,  363 

Alcohols,  higher,  fermentation  of,  75 
Aleppo  boil,  568 
Alexines,  478,  500 
Amboceptors,  480,  491 
Amoebic  dysentery,  537 
Amcebulse  of  malaria,  523 
Anaerobes,  17 
cultures  of,  60 
separation  of,  57 
toxins  of,  60 

Anaerobic  Esmarch's  tubes,  60 
Anaerobic  fermentation  tubes,  60 
Anaerobic   plate   cultures,   Bulloch's 

apparatus  for,  58 
Anaesthetic  leprosy,  269 
Aniline  oil,  dehydrating  by,  93 

water,  98 

Aniline  stains,  list  of,  94 
Animals,  autopsies  on,  123 

inoculation  of,  120 
Anthrax,  300 
anti-serum,  315 
bacillus,  301 
biology  of,  304 
cultivation  of,  302 
inoculation  with,  308 
toxins  of,    312 


594 


INDEX 


Anthrax,  diagnosis  of,  317 

in  animals,  306 

in  man,  309 

protective  inoculation,  314 

spread  of,  313 
Anti-abrin,  470 
Anti-anthrax  serurn,  315 
Anti-bacterial  sera,  476 

properties  of,  477 
Anti-cholera  vaccination,  412 
Anti-diphtheritic  serum,  467 
Antikorper,  457 
Anti-plague  inoculation,  436 
Anti-plague  sera,  436 
Antipneumococcic  serum,  210 
Antirabic  serum,  519 
Anti-ricin,  470 
Antiseptics,  141 

actions  of,  144 

standardisation  of,  143 

testing  of,  142 
Antisera,      therapeutic     action     of, 

488 

Autistreptococcic  serum,  489 
Antitetanic  serum,  384 

preparation  of,  467  et  seq. 
Antitoxic  action,  nature  of,  470 

bodies  in  normal  tissues,  474 

sera,  use  of,  469 

serum,  467 
cholera,  411 
standardisation  of,  468 
Antitoxins,  chemical  nature  of,  470 

origin  of,  474 
Antitubercular  serum,  264 
Antityphoid  serum,  344 
Appendicitus,  185 
Arthrospores,  question  of  occurrence 

of,  7 
Artificial    immunity,     varieties     of, 

457  et  seq. 

Attenuation  of  virulence,  457 
Autoclave,  29 
Autolysis  of  bacteria,  162 
Autopsies  on  animals,  123 
Avian  tuberculosis,  250 

Bacilli,  acid-fast,  239,  252 
stain  for,  100 

characters  of,  12 
Bacillus  acidi  lactici,  20 

aerogenes  capsulatus,  188,  397 

anthracis,  301 

botulinus,  393 


Bacillus  coli  communis,  lesions  caused 
by,  183  et  seq. 

characters  of,  324 

comparison  with  b.  typhosus,  325 
coli  in  soil,  133,  134 

pathogenicity  of,  333 
diphtherise,  353 
dysenterise  Shiga-Flexner,  346 
enteritidis  (Gaertner),  336 
enteritidis  sporogenes,  350 

in  soil,  133,  134 
of  glanders,  277 
icteroides,  452 
of  influenza,  420 
Koch-Weeks,  191 
lactis  aerogenes,  180 
lacunatus,  193 
of  leprosy,  269 
of  malignant  oedema,  390 
Miiller's,  192 
mycoides  in  soil,  132 
ozcenae,  285 
paratyphosus,  335 
of  plague,  426 
pneumonise,  199 
pseudo-diphthericus,  366 
of  psittacosis,  337 
pyocyaneus,  180 

agglutination  of,  485 

occurrence  of,  185 
pyogenes  fcetidus,  174 
of  quarter-evil,  396 
of  rhinoscleroma,  284 
of  smegma,  254 
of  soft  sore,  227 
subtilis,  57 
of  syphilis,  229 
tetani,  372 
of  Timothy  grass,  253 
of  tubercle,  237 
of  typhoid,  319 

differentiation  from  b.  coli,  325 
of  xerosis,  367 

Bacteria,  action  of  dead,  154 
aerobic  (v.  Aerobes),  17 
anaerobic  (v.  Anaerobes),  17 
biology  of,  16 
capsulated,  3 
chemical  action  of,  21 

composition  of,  9 
classification  of,  11 
cultivation  of,  25 
death  of,  141 
effects  of  light  on,  19 


INDEX 


595 


Bacteria,  food  supply  of,  16 

higher,  14 

lower,  11 

microscopic  examination  of,  85 

morphological  relations  of,  2 

motility  of,  7 

movements  of,  19 

multiplication  of,  4 

nitrifying,  23 

parasitic,  21 

pathogenic,  action  of,  149 
effects  of,  155 

saprophytic,  21 

separation  of,  51 

species  of,  23 

spore  formation  in  (v.  also  Spores), 
5,  56 

structure  of,  3 

sulphur-containing,  9 

temperature  of  growth  of,  18 

toxins  of,  161 

variability  among,  23 

virulence  of,  150,  461 
Bacterial  ferments,  22,  168 

pigments,  10 

protoplasm,  structure  of,  8 

treatment  of  sewage,  139 
Bactericidal  powers  of  serum,  477 

substances,  477 
Bacteriological  diagnosis,  118 

examination  of  discharges,  116 
Beer  wort  agar,  44 
Beggiatoa,  15 

Behring  on- immunity,  385,  468 
Bile-salt  media,  43 
Bismarck-brown,  95 
Blackleg,  396 
Blackwater  fever,  534 
Blastophores  (malaria),  529 
Blood-agar  (v.  also  Culture  media), 

38 
Blood,  examination  of,  68,  88 

in  malarial  fever,  521 

in  relapsing  fever,  438 

serum,  coagulated,  as  medium,  40 
Bone-marrow  in  leucocytosis,  156 
Bordet's  phenomenon,  477 
Botulism,  bacillus  of,  393 
Bouillon  (v.  also  Culture  media),  32 
Bovine  tuberculosis,  248 
Bread  paste,  47 
Briegerand  Boer,  166 

Fraenkel,  162 
Buboes,  227 


Bubonic  pest,  429 

Buchner  on  alexines,  500 

Bulloch's    apparatus    for    anaerobic 

culture,  58 

Biitschli  on  bacterial  structure,  9 
Butter  bacilli,  acid  fast,  253 

Calmette,  435,  462,  469,  471 
Canon  on  influenza,  420 
Cantani  on  influenza,  424 
Capaldi  and  Proskauer,  media  of,  328 
Capsules,  staining  of,  102 
Carbol-fuchsin,  99 

-methylene-blue,  98 

-thionin-blue,  98 
Carbolic  acid  as  antiseptic,  147 
Carroll's  method  of  making  anaerobic 

cultures,  60 

Carter  on  relapsing  fever,  440 
Cattle  plague,  507 
Cerebro- spinal  fluid,  examination  by 

lumbar  puncture,  68 
Chamber-land  and  Koux,  attenuation 

of  b.  anthracis,  460 
Chamberland's  filter,  70 
Chemiotaxis,  20,  495 
Chlorine  as  antiseptic,  144 
Cholera,  399 

immunity  against,  410 

inoculation  of  man  with,  410 

methods  of  diagnosis  of,  412 

preventive  inoculation  against,  412 

-red  reaction,  404 
Cholera  spirillum,  400 
distribution  of,  402 
inoculation  with,  406 
powers  of  resistance  of,  405 
relations  to  disease,  414 
toxins  of,  408 
Cladothrices  in  soil,  132 
Cladothrix,  15 

asteroides,  295 
Clubs  in  actinomyces,  289 
Cocci,  characters  of,  11 
Collodion    capsules,   preparation   of, 

123 

Colonies,  counting  of,  65 
Comma  bacillus,  399 
Commission  on  tuberculosis,  248 

vaccination,  505 
Complement,  478 

deviation  of,  488 
Conjunctivitis,  191 
Conradi-Drigalski  medium,  42 


596 


INDEX 


Copeman  on  smallpox,  508 
Cornet's  forceps,  87 
Corrosive  films  of  blood,  etc.,  89 
Corrosive  sublimate,  as  antiseptic,  145 

fixing  by,  90 
Councilman  and  Lafleur  on  dysentery, 

537 
Counting  of  colonies,  65 

dead  bacteria  in  a  ciilture,  67 
living  bacteria  in  a  culture,  66 
Cover-glasses,  cleaning  of,  87 
Cowpox,  relation  to  smallpox,  505 
Crescentic  bodies  in  malaria,  523 
Cultivation  of  anaerobes,  57 
Culture  media,  preparation  of:  agar, 

36 

alkaline  blood  serum,  41 
blood  agar,  38 

serum,  39 
bouillon,  32 
bread  paste,  47 
glucose  agar,  37 
broth,  32 
gelatin,  35 
glycerin  agar,  37 

broth,  35 
litmus  whey,  44 
Loffler's  serum  medium,  40 
Marmorek's  serum  media,  41 
meat  extract,  31 
peptone  gelatin,  35 

solution,  38 
potatoes,  44 
serum  agar,  38 
Cultures,  destruction  ot,  83 
filtration  of,  69 
from  organs,  117,  124 
hanging-drop,  aerobic,  63 

anaerobic,  64 
incubation  of,  79 
microscopic  examination  of,  86 
permanent  preservation  of,  82 
plate,  52j 
pure,  48 
"shake,"  77 
Cutting  of  sections,  92 
Cystitis,  185,  224 
Cytases,  478,  496 
Cytolytic  sera,  482 

De  Bary,  definition  of  species,  23 
Decolorising  agents,  97 
Deep  cultures,  60 
Delhi  sore,  568 


Dehydration  of  sections,  93 
Delepine,  110 
Deneke's  spirillum,  419 
Dextrose- free  bouillon,  75 
Diagnosis,  bacteriological,  115,  118 
Diphtheria,  352 
diagnosis  of,  368 
immunity  against,  467 
origin  and  spread  of,  365 
paralysis  in,  353,  361 
results  of  treatment,  488 
Diphtheria  bacillus,  action  of,  358 
bacilli  allied  to,  365 
characters  of,  353 
distribution  of,  354 
inoculation  with,  359 
isolation  of,  368 
powers  of  resistance  of,  359 
staining  of,  108,  359 
toxins  of,  163,  361 
variations  in  virulence  of,  364 
Diplo-bacillus  of  conjunctivitis,  193 
Diplococcus,  12 
catarrhalis,  217 
crassus,  217 

endocarditidis  encapsulatus,  188 
intracellularis  meningitidis,  213 
pneumonias,  199 
Disturbances      of      metabolism     by 

bacteria,  159 

Drigalski-Conradi  medium,  42 
Drying  of  sera,  etc.,  in  vacuo,  78 
Ducrey's  bacillus,  227 
cultivation  of,  228 
Dum-Dum  fever,  563 
Durham's  fermentation  tubes,  76 
Dysentery,  amoebic,  537 
bacteria  in,  346 
characters  of  amoeba  of,  537 
Dysentery,   methods   of  examination 
in,  347,  542 

East  coast  fever  in  cattle,  569 

Eberth's  bacillus,  319 

Ehrlich  on  ricin  and  abrin,  464,  469 

on  toxins,  170 

side-chain  theory  of  antitoxin  for- 
mation, 491 
Eisner's  medium,  46 
Embedding  in  paraffin,  91 
Empyema,  205,  423 
Endocarditis,  bacteria  in,  188 
Enheemosphores  (malaria),  523 
Entamoaba  coli,  537 


INDEX 


597 


Eutamoeba  histolytica,  537 

cultivation  of,  539 
Euteric  fever,  319 
Enteritis,  dysenteric,  347,  540 
Epidemic    cerebrospinal    meningitis, 

213 

Eppingev's  streptothrix,  295 
Ermengem  on  botulism,  394 

stain  for  flagella,  104 
Erysipelas,  191 
Escherich's  bacillus,  319 
Esmarch's  roll-tubes,  55,  59 

anaerobic,  60 

Exaltation  of  virulence,  461 
Examination  of  water,  135 
Exhaust-pump,  70 
Exotospores  (malaria),  522 


False  membrane,  184,  353 
Farcy,  276 

Feeding,  immunity  by,  464 
Fermentation    by    pueunio  -  bacillus, 
204 

by  bacillus  coli,  326 

methods  of  observing,  74 

of  sugars  by  bacteria,  74 

test  of  bacterial  action,  74 

tubes,  76 

anaerobic,  60 

Ferments   formed    by   bacteria,    22, 
168 

in  diphtheria,  364 

in  tetanus,  380 
Fever,  159 
Film  preparations,  dry,  86 

wet,  88 

staining  of,  95 

Filter,  porcelain,  gelatined,  166 
Filtration  of  cultures,  69 
Finkler  and  Prior's  spirillum,  418 
Fish,  tuberculosis  in,  251 
Fixateurs,  497 
Fixation  of  tissues,  89 
Flagella,  nature  of,  8 

staining  of,  103 

Flagellated  organisms  in  malaria,  528 
Flligge,  14 

Forceps  for  cover-glasses,  87 
Formalin  as  antiseptic,  146 
Foth's  dry  mallein,  283 
Fraenkel's  pneumococcus,  192,   198, 
199 

stain  for  tubercle,  101 


Framboesia,  spirochaetes  in,  234 
Frankland,  on  water  bacteria,  137 
Eraser,  T.  K.,  462,  469,  475 
Friedlander's    pneumobacillus,    198, 

203 

Frisch  on  rhinoscleroma,  284 
Fuchsin,  carbol-,  99 

Gamaleia  on  pneumonia,  206 
Gametocytes  (malaria),  527 
Gangrenous  emphysema,  389,  392 

pneumonia,  423 

Gas  formation,  observation  of,  44,  76 
Gas -regulator,  80 
Geissler's  exhaust-pump,  70 
Gelatin  media,  35 

phenolated,  345 

separation  by,  51 
Gelatined  porcelain  filter,  166 
Gentian-violet,  98 
Germicides,  141 
Geryk  pump,  78 
Giemsa's  stain,  107 

stain  for  spirochsetes  in  films,  107 
Glanders,  275 

diagnosis  of,  283 

in  horses,  276 

in  man,  276 

lesions  in,  281 
Glanders  bacillus,  277 
agglutination  of,  282 
inoculation  with,  280 
Glossina  morsitans,  552 

palpalis,  559 
Glucose  media,  35  et  seq. 
Glucosides,  fermentation  of,  75 
Glycerin  media,  35  et  seq. 

potato  as  culture  medium,  46 
Golgi  on  malaria,  521 
Gonidia,  15 
Gonococcus,  characters  of,  219 

inoculation  with,  222 

toxin  of,  223 
Gonorrhoea,  219 
Gonorrhceal  conjunctivitis,  225 

endocarditis,  225 

septicaemia,  226 
Gram's  method,  99 

Weigert's  modification  of,  100 
Grease,  504 

Greenfield  on  anthrax,  311,  446,  460 
Griiber  and  Durham's   phenomenon, 

485 
Guarnieri  bodies  in  smallpox,  508 


598 


INDEX 


Gulland  (methods),  89,  92 
Hsemamoeba  Danilewski,  530 
malarise,  530 
prsecox,  530 

relicta,  530 

vivax,  530 

Hsematozoon  malarise,  520 
Hsemolytic  sera,  479 
Hsemolytic  tests,  methods  of,  483 
Haffkine    on  anti  -  cholera    inocula- 
tion, 412 
Haffkine's  inoculation  method  against 

plague,  436 
Halteridium,  528,  530 
Hanging-drop  cultures,  63 

examination  of,  85 
Hankin,  312 

Hansen,  leprosy  bacilli,  269 
Hesse's  tube,  127 
Hiss's  serum  water  media,  41 
Hofmann's  bacillus,  366 
Horsepox,  504 

Houston  on  bacteriology  of  soil,  1 31 
Hueppe,  7,  14 
Hydrogen,  supply  of,  58 
Hydrophobia,  510 

diagnosis  of,  519 

Negri  bodies  in,  514 

prophylactic  treatment  of,  516 

the  virus  of,  513 
Hypodermic  syringes,  121 

Immune-bodies,  478 

origin  of,  482 

Immunity  (v.  also  Special  Diseases), 
456 

acquired,  theories  of,  490 

active,  458,  459 

artificial,  457 

by  feeding,  464 

by  toxins,  462 

methods,  459 

natural,  498 

passive,  458,  464 

unit  of,  468 

Impression  preparations,  118 
Incubators,  79 
Indol,  formation  of,  77 
Infection,  conditions  modifying,  149 

nature  of,  153 
Inflammatory     conditions      due     to 

bacteria,  157 
Influenza,  420 

bacilli,  pseudo-,  423 


Influenza,  bacillus,  cultivation  of,  421 
bacillus,  inoculation,  424 
lesions  in,  422 
sputum  in,  422 
Inoculation,  methods  of,  120 
of  animals,  120 

separation  by,  56 
protective,  462  et  seq. 
Intestinal  changes  in  cholera,  402 
amcebic  dysentery,  539 
bacterial  dysentery,  347 
typhoid  fever,  329 

Intestinal   infection   in   cholera   (ex- 
perimental), 407 
Involution  forms  in  bacteria,  4 
Iodine  solution,  Gram's,  99 
terchloride,  468 

as  antiseptic,  145 
lodoform  as  antiseptic,  148 
Issaeff,  464 
Ivanoff's  vibrio,  415 

Japanese  dysentery,  350 
Jenner  on  vaccination,  503 
Jenner's  stain,  106 
Johne's  bacillus,  254 
Joints,  gonococci  in,  225 

Kala-azar,  563 
Kipp's  apparatus,  58 
Kitasato  on  bacillus  -of  influenza,  420 
of  plague,  425 
of  tetanus,  372  et  seq. 
Klebs-Loffler  bacillus,  352 
Klein,  345,  508 
Klemperer  on  pneumonia,  210 
Koch  on  avian  tuberculosis,  250 

bacillus  of  malignant  oedema,  388 

bovine  tuberculosis,  248 

cholera  spirillum,  399 

cultivation  of  b.  anthracis,  301 

leveller  for  plates,  53 

tubercle  bacillus,  235 

tuberculin,  258 

"tuberculin  0,"  and  "R,"  260 
Koch- Weeks  bacillus,  191 
Korn's  acid-fast  bacillus,  253 
Kruse  and  Pasquale  on  dysentery,  541 
Kubel-Tiemann  litmus  solution,  42 
Kiihne's  methylene-blue,  98 

modification  of  Gram's  method,  100 
Lamb  on  relapsing  fever,  442 
Laveran's  malarial  parasite,  521 
Leishman- Donovan  bodies,  563 


INDEX 


599 


Leishman-Donovan  bodies,  cultivation 

of,  566 

Irishman's  opsonic  technique,  111 
serum    method   for    staining    try- 

panosomes,  545 
stain,  106 
Leishmania  donovaui,  567 

tropica,  567,  568 
Lenses,  85 
Lepra  cells,  269 
Leprosy,  267 
bacillus,  269 

distribution  of,  271 
staining,  100,  270 
diagnosis  of,  274 
etiology  of,  272 

Leprosy-like  disease  in  rats,  273 
Leptothrix,  15 

Lesions  produced  by  bacteria,  155 
Leucocidin,  165 
Leucocytosis,  156,  495 
Leucomaines,  161 
Levaditi's  method  for  staining  spiro- 

chaetes,  104 
Litmus  solution,  Kubel-Tiemann's,  42 

whey,  44 

Liver  abscess  in  dysentery,  540 
Lockjaw,  371 
Ldffler's  bacillus,  352 
methylene-blue,  98 
sea-urn  medium,  40 
and  Schutz'  glanders  bacillus,  275 
Losch,  amoeba  of,  537 
Lumbar  puncture,  68 
Lustgarten's  bacillus,  229 
Lustig's  anti-plague  serum,  436 
Lymph,  vaccine,  506 
Lymphangitis,  184 
Lysogenic  action  of  serum,  477 
towards  blood  corpuscles,  479 

MacCoukey's  bile-salt  media,  43 
medium,  use  of  in  dysentery,  347 
in  examining  water,  136 
in  paratyphoid  fever,  335 
M'Fadyean  on  glanders,  282 
Macrocytase,  497 
Macrophages,  495 
Madura  disease,  297 
Malaria,  cycle  in  man,  522 

in  mosquito,  528 
pathology  of,  533 
prevention  of,  532 
question  of  immunity  against,  534 


Malarial  fever,  examination  of  blood 

in,  535 

malignant,  523,  531 
mosquitoes  in,  532 
Malarial  parasite,  521 
inoculation  of,  522 
staining  of,  Leishman's  method, 
106 

Romanowsky  methods,  106 
varieties  of,  529 

Malignant  oedema,  bacillus  of,  388 
diagnosis  of,  393 
immunity  against,  393 
Malignant  pustule,  310 
Mallein,  283 
Malta  fever,  446 

methods  of  diagnosis,  450 
spread  of  disease,  449 
Mann's  method  of  fixing  sections,  92 
Manson,  521 
Maragliauo's  anti- tubercular  serum 

264 
Marchiafava   and    Celli   on    malaria 

521 

Marmorek,  on  streptococci,  183 
antistreptococcic  serum,  476 
Marmorek's  serum  media,  41 
antitubercular  serum,  265 
Martin,  Sidney,  on  albumoses,  etc.,  165 
on  anthrax,  312 
on  diphtheria,  363 
Martin,  C.  J.,  on  toxins,  166 

on  antitoxins,  475 
Massowah  vibrio,  416 
Measuring  bacteria,  119 
Meat  extract,  31 
Meat-poisoning  by  bacillus  botulinus, 

393 

by  Gaertuer's  bacillus,  336 
Mediterranean  fever,  446 
Meningitis,  bacteria  in,  217 

epidemic  cerebro-spinal,  174,  213 
in  influenza,  423 
pneumococci  in,  205 
posterior  basal,  216 
Mercury  perchloride  as  antiseptic,  145 
Metabolism,     disturbances     of,     by 

bacteria,  159 

Metachromatic  granules,  8 
Metchnikoff  on  cholera  in  rabbits,  407 

relapsing  fever,  441 
Metchnikoff  s  phagocytosis  theory,  495 

spirillum,  417 
Methylene-blue,  95,  98 


600 


INDEX 


Methyl-violet,  94 

Meyer  and  Ransom  on  tetanus  toxin, 

382 

Micrococci  of  suppuration,  174 
Micrococcus,  12 

of  gonorrhoea,  219 

meliteusis,  447 

pyogenes  tenuis,  174 

tetragenus,  181 

lesions  caused  by,  185 

ureae,  20 

Microcytase,  497 
Microphages,  495 
Microscope,  use  of,  85 
Microtomes,  90 
Migula,  12 

Mikulicz,  cells  of,  284 
Milk  as  culture  medium,  46 
Moller's  stain  for  spores,  102 
Moeller's  Timothy -grass  bacillus,  253 
Morax,  bacillus  of,  192,  193 
Mordants,  97 

Morgan's  bacillus,  No.  1,  351 
Mosquitoes,  in  malaria,  528,  532 

r61e  in  yellow  fever,  453 
Moulds,  media  for  growing,  44 
Muencke's  filter,  72 
Miiller's  bacillus,  192 
Mycetoma,  297 
Myelocytes,  neutrophile,  156 

Nagana,  552 

Natural  immunity,  498 

Neelsen's  stain  for  tubercle,  101 

Negative  phase  in  immunisation,  262, 

494 

Negri  bodies  in  rabies,  514 
Neisser's  gonococcus,  219 

stain  for  b.  diphtherias,  108 
Neiicki,  10 

Neutral-red  as  indicator  for  media,  43 
use  of,  38 

with  b.  typhosus,  327 
Neutrophile  leucocytes,  156 

myelocytes,  156 
Nicolaier,  tetanus  bacillus,  371 
Nicolle's     modification     of     Gram's 

method,  100 

Nikati  and  Rietsch  on  cholera,  407 
Nitrifying  bacteria,  23 
Nitroso-indol  body,  78 
Nordhafen  vibrio,  418 
Novy    and    MacNeal,    medium    for 

culture  of  trypanosomes,  546 


Obermeier's  spirillum,  438 

(Edema,  malignant,  388 

Ogata's  dysentery  bacillus,  350 

Ogston,  174 

Oil,  aniline,  for  dehydrating,  etc.,  93 

Oil  immersion  lens,  85 

Ookinete,  528,  547,  548 

Opsonic  action,  nature  of,  483 

technique,  111 
Opsonius,  112 

absorption  of,  484 

in  tuberculosis,  261 

thermolabile,  484 

thermostable,  484 

Organisms  lower  than  bacteria,  2,  452 
Oriental  plague,  425 
Osteomyelitis,  190 
Otitis,  205,  423 

Oxygen,  nascent,  as  antiseptic,  145 
Ozoena  bacillus,  285 

Para-colon  bacillus,  335 
Paraffin  embedding,  91 
Paratyphoid  bacillus,  335 
Passage,  461 

Passive  immunity,  458,  464 
Pasteur  on  exaltation  of  virulence  of 
bacteria,  461 

on  hydrophobia,  516 

on  vaccination  against  anthrax,  314 

septicemie  de,  388 
Pathogenicity  of  bacteria,  149 
Peptone  gelatin  (v.  Culture  media),  35 

solution,  38,  404 

Periostitis,  acute  suppurative,  190 
Peritonitis,  184,  224 
Perlsucht,  236 
Pestis  major,  431 

minor,  431 
Petri's  acid-fast  bacillus,  253 

capsules,  52 

sand-filter  for  examining  air,  128 
Petruschky's  litmus  whey,  44 
Petteukofer  on  cholera,  410,  415 
Pfeffer,  20 
Pfeiffer  on  anti-serum,  477 

cholera,  411 

influenza,  420 

typhoid,  333 

Pfeiffer's  phenomenon,  411,  477 
Phagocytes,  156 
Phagocytosis  theory  of  Metchnikoff, 

495 
Phenol  broth,  347 


INDEX 


601 


Phenol-phthaleiu  as  indicator,  33 
Phenomenon  of  Bordet,  477 
Griiber  and  Durham,  485 
Pfeiffer,  411,  477 
Pigments,  bacterial,  10 
Pipettes,  66,  108,  110,  116 
Piroplasmata  as  causes  of  disease,  569 
Piroplasmosis,  568 
Pitfield's  flagella  stain,  103 
Plague,  bacillus  of,  426  et  seq, 

Haff  kine's  inoculation  against,  436 

immunity  against,  435 

infection  in,  432 

involution  forms,  427 

part   played   by   rat   fleas   in   the 

spread  of,  433 

preventive  inoculation  against,  436 
serum  diagnosis,  437 
stalactite  growths  of,  429 
varieties  of,  431 
Plasmolysis,  9 
Plate  cultures,  agar,  55 
gelatin,  51 
gouococcus,  222 
Platinum  needles,  49 
Pneumobacillus  (Friedliiuder's),  199, 

203  et  seq. 
Pneumococcus      (Fraenkel's),      199, 

201  et  seq. 

immunity  against,  210 
in  endocarditis,  188 
lesions  caused  by,  204 
toxins  of,  209 

Pneumonia,  bacteria  in,  197 
gangrenous,  423 
in  influenza,  422 
methods  of  examination  of,  212 
septic,  197 
varieties  of,  196 
Polar  granules,  8 
Positive  phase  in  immunisation,  262, 

494 
Potassium  permanganate  as  antiseptic, 

147 

Potatoes  as  culture  material,  44 
Poynton     and      Payne     on      acute 

rheumatism,  193 
Precipitins,  487 
Preparations,  impression,  118 
Protective  inoculation,  462  et  seq. 
Proteosoma,  530 
Protozoa  described   in  hydrophobia, 

513 
smallpox,  508 


Protozoon  malariae,  521 
Pseudo- diphtheria  bacillus,  365 

-tuberculosis  streptothricea,  296 
Psittacosis  bacillus,  337 
Ptomaines,  161 
Puerperal  septicaemia,  184 
Pus,  examination  of,  87,  195 
Pustule,  malignant,  310 
Pyaemia,  184  et  seq. 

nature  of,  173 

Quartan  fever,  530 
Quarter-evil,  bacillus  of,  396 
Quotidian  fever,  529 

Kabies,  510 

Rabinowitch's  acid-fast  bacillus,  253 

Rauschbrand  bacillus,  396 

Ray-fungus  (actinomyces),  286 

Reaction  of  media,  standardising  of, 
33 

Receptors,  491 

Recovery  from  disease,  457 

Red  stains,  95 

Red-water  fever  in  cattle,  569 

Reichert's  gas  regulator,  80 

Relapsing     fever,     agglutination     of 

spirillum,  442 
bactericidal  serum  in,  442 
spirillum  of,  etc.,  439 

Reversibility  of  toxin-antitoxin  reac- 
tion, 472 

Rheumatism,  acute,  193 

Rhinoscleroma,  bacillus  of,  284 

Ricin,  169 

immunity  against,  464,  469 

Rivers,  bacteria  in,  137 

Robin,  169 

Rock  fever,  446 

Roll-tubes,  Esmarch's,  55,  59 

Romanowsky  stains,  105 

Rosenbach  (bacteria  in  suppuration), 
174 

Ross,  on  malaria,  521 

thick    film    method    for   malarial 
parasite,  536 

Roux  on  antitoxic  sera,  469 

and  Yersin  (diphtheria),  361  et  seq. 

Sabouraud's  medium,  44 
Safranin,  95 

Salt-agar  as  medium  for  b.  pestis,  427 
Sanarelli  (typhoid  fever),  332 
Sanderson,  Burdon,  460,  507 


602 


INDEX 


Saprophytes,  149 

Sarcina,  12 

Sausage  poisoning,  bacillus  botulinus 

in,  394 

Schaudiuu  on  biology  of  trypauosomes, 
548 

on  amoebae  of  dysentery,  537 

on  morphology  of  spirilla,  550 

on  spirochsete  pallida,  229 

on  spirillum  Ziemanni,  550 
Schizomycetes,  3 
Schizophyceae,  3 
Schizophyta,  3 
Schiiffner's  dots,  106,  531 
Sclavo's  anti-anthrax  serum,  315 
Scorpion  poison,  169 
Section-cutting,  90 
Sections,  dehydration  of,  93 
Sedimentation  methods,  109 

test  for  typhoid,  338 
Seiteiiketten,  491 
Septicaemia,  nature  of,  173 

puerperal,  184 

sputum,  197 

Septicemie  de  Pasteur,  388 
Septic  pneumonia,  197 
Sera,  haemolytic,  479 
Serum  agar.  38 
Serum,  agglutinative  action  of,  485 

anaphylaxis,  494 

antibacterial,  476 

an ti- cholera,  411 

antidiphtheritic,  467 

anti-plague,  436 

antipueumococcic,  210 

antirabic,  519 

antistreptococcic,  476 

antitetanic,  384 

antitoxic,  preparation  of,  467  et  seq. 

antitubercular,  264 

antityphoid,  344 

bactericidal  action  of,  477 

blood  (v.  Culture  media),  39 

diagnosis,  485 
methods,  109 
of  typhoid,  337 

inspissator,  39 

lysogenic  action  of,  477 

towards  blood  corpuscles,  479 
Serum  media,  39 
Serum- water  media,  41 
Sewage,  bacterial  treatment  of,  139 

contamination  of  water  by,  136 
Shake  cultures,  77 


Sheep-pox,  507 

Shiga's  bacillus,  346 

Side-chain  theory,  Ehrlich's,  491 

Sleeping  sickness,  555 

Slides  for  hanging-drops,  63,  64 

Sloped  cultures,  aerobic,  48 

anaerobic,  62 
Smallpox,  503 
bacteria  in,  507 
Guarnieri  bodies  in,  508 
Smegma  bacillus,  254 
Smith's,  Lorrain,  serum  medium,  41 
Smith,    Theobald,     phenomenon   of, 

494 
Snake  poisons,  169 

activating  of,  by  serum,  170 
constituents  of,  169 
immunity  against,  462 
Sobernheim's  anti-anthrax  serum,  316 
Soft  sore,  227 

Soil,  examination  of,  for  bacteria,  131 
Soudakewitch  on  relapsing  fever,  442 
Spinal  cord,  lesions  by  pyogenic 

organisms,  183 
Spirilla,  characters  of  (v.  also  Vibrio), 

14,  550 

like  cholera  spirillum,  417 
Spirillosis  in  animals,  439 
Spirillum  Metchuikovi,  417 
of  cholera,  400 
Deneke,  419 
Finkler  and  Prior,  418 
Miller,  419 
relapsing  fever,   inoculation  with, 

etc.,  438 

Spirochaete,  14,  229,  550 
pallida,  229 

staining  of,  104,  107 
pertenuis,  234 
refringens,  230 
Spirochsetes  in  syphilis,  229 
in  yaws,  234 
staining  of,  in  films,  107 
staining  of,  in  sections,  104 
Spironema  pallidum,  229 
Splenic  fever,  300 
Spore  formation,  arthrosporous,  7 
endogenous,  5 
in  b.  anthracis,  304 
Spores,  staining  of,  102 
Sporoblasts,  529 
Sporocyst  (malaria),  529 
Sporocytes,  in  malaria,  524 
Sporozoites,  529 


INDEX 


603 


Sporulation  of  malarial  parasite,  522 
Sputum,  amoebae  in,  541 
influenza,  422,  425 
in  plague,  238 
in  pneumonia,  200 
phthisical,  241,  255,  265 
septicaemia,  197 
Staining  methods,  94  et  seq. 

of  capsules,  Welch's  method,  102 

Kichard  Muir's  method,  102 
of  flagella,  103 
of  leprosy  bacilli,  270 
of  spores,  102 
of  tubercle  bacilli,  100 
principles,  94 
Stains,  basic  aniline,  94 
Standard  of  immunity,  468 
Standardising  reaction  of  media,  33 
Staphylococci,  lesions  caused  by,  184 
Staphylococcus,  12 
cereus  albus,  176 

flavus,  176 
pyogenes  albus,  176 

aureus,  characters  of,  174 

inoculation  with,  182 
citreus,  174 

Steam  steriliser,  Koch's,  27 
Stegomyia  fasciata,  453 
Sterilisation  by  heat,  26  et  seq. 
at  low  temperatures,  29 
by  steam  at  high  pressure,  29 
Streptococci  in  diphtheria,  356 
in  false  membrane,  184 
lesions  caused  by,  184 
varieties  of,  178 
Streptococcus,  12 
anginosus,  179 
brevis,  178 

conglomeratus,  178,  179 
equinus,  179 
erysipelatis,  191 
fsecalis,  179 
longus,  178 
mitis,  179 
pneumoniae,  198 
pyogenes,  characters  of,  176 
inoculation  with,  182 
in  air,  130 
in  soil,  133 
salivarius,  179 
Streptothrices  allied  to  actinomyces, 

294 

Streptothrix,  15 
actinomyces,  287 


Streptothrix,    anaerobic     in    actino- 
mycosis,  293 

madurae,  297 
Subcultures,  49 
Sugars,  classification  of,  74 

fermentation  of,  74 
Sulphurous  acid  as  antiseptic,  147 
Summer  diarrhoea,  bacteria  in,  351 
Suppuration,  bacteria  of,  174 

gonococci  in,  223 

methods  of  examination  of,  195 

nature  of,  172 

origin  of,  186 

pueumococci  in,  205 

typhoid  bacillus  in,  330 
Symptoms  caused  by  bacteria,  161 
Syphilis,  bacillus  of,  229 

spirochsete  pallida  in,  229 

transmission  to  animals,  233 
Syringes  for  inoculation,  120,  121 

Tabes  mesenterica,  257 
Taurocholate  media,  43 
Tertian  fever,  530,  531 
Test-tubes  for  cultures,  47 
Tetanolysin,  380 
Tetanospasmin,  380 
Tetanus,  371 

anti-serum  of,  384,  467  et  seq. 
intravenous  injection  of,  386 

cerebral,  384 

dolorosus,  383 

immunity  against,  384        ^ 

methods  of  examination  in,  388 

treatment  of,  386,  489 
Tetanus  bacillus,  372 

inoculation  with,  378 
isolation  of,  373 
spores  of,  373 
toxins  of,  163,  379 
Tetrads,  12 
Texas  fever,  569 
Theory  of  exhaustion,  490 

of  phagocytosis,  495 

of  retention,  490 

humoral,  490 
Thermophilic  bacteria,  18 
Thermostable  opsonins,  484 
Thionin-blue,  95,  98 
Thiothrix,  15 
Tick  fever,  African,  443 
Timothy-grass  bacillus,  153 
Tissues,  action  of  bacteria  on,  155 

fixation  of,  89 


604 


INDEX 


Tizzoni  and  Cattani  on  tetanus,  385 
Toxalbumius,  162 
Toxic  action,  theory  of,  170 
Toxicity,  estimation  of,  467 
Toxins,  concentrated,  method  of  ob- 
taining, 167 
constitution  of,  491 
early  work  on,  161 
effects  of,  158 
immunisation  by,  467 
intra-  and  extra-cellular,  162 
nature  of,  165 
non-proteid,  166 
of    anthrax,     cholera,     etc.    (vide 

Special  Diseases) 
production,  154 
susceptibility  to,  491 
vegetable,  169 
Toxoids,  171,  472 
Toxones,  171 

Trachoma,  bacteria  in,  192,  424 
Trichophyta,  media  for  growing,  44 
Tropical  ulcer,  568 
Trypanosoma  gambiense,  558 
Lewisi,  544,  551 
noctuse,  548 

of  sleeping  sickness,  555 
ugandense,  544,  558 
ugandense,    relation  to   Tr.    Gam- 
biense, 561 
Trypanosomata       associated       with 

various  diseases,  544 
culture  of,  546 
morphology  of,  544 
sexual  cycle  in,  548 
Trypanosomiasis,  544 
Tse-tse  fly  disease,  552 
Tubercle  bacillus,  237 
action  of  dead,  255 
avian,  250 
cultivation  of,  239 
distribution  of,  243 
immunity  against,  260 
inoculation  with,  246 
powers  of  resistance  of,  241 
in  sputum,  etc.,  255,  265 
toxins  of,  258 
stains  for,  100,  239 
giant  cells,  242 

methods  of  examination  of,  265 
Tubercles,  structure  of,  242 
Tubercular  leprosy,  268 
Tuberculin,  258 

"Bazillenemulsion."  260 


Tuberculin,  "0"  and  "R,"  260 
Tuberculosis,  235 
aviau,  250 
bovine,  248 

its  relation  to  human,  248 
diagnosis  by  tuberculin,  259 
Tuberculosis,  in  animals,  236 
in  fish,  251 

modes  of  infection,  256 
precautions  in  diagnosis  of,  255 
Tubes,  cultures  in,  47 
Typhoid  bacillus,  319 

comparison  with  b.  coli,  325 
examination  for,  344 
immunity  against,  332 
inoculation  with,  331 
isolation  from  water  supplies,  345 
toxins  of,  332 
serum  diagnosis,  337 
suppurations  in,  330 
vaccination  against,  343 
Typhoid  fever,  319 

pathological  changes  in,  329 

Ulcerative  endocarditis,  188 
experimental,  190 
gonococci  in,  225 
Unit  of  immunity,  468 
Urine,  examination  of,  69 
staining  of  bacteria  in,  88 
tubercle  bacilli  in,  246,  265 
typhoid  bacilli  in,  344 

Vaccination  against  smallpox,  502 

against  hydrophobia,  516 

against  typhoid,  343 

for  infection  by  pyogenic  bacteria, 
'  194 

nature  of,  509 
Variola,  505  et  seq. 
Veuins,  169 
Vibrio  (see  also  Spirillum),  14 

berolinensis,  415 

of  cholera,  400 

Danubicus,  415 

Deneke's,  419 

Finkler  and  Prior's,  418 

Gindha,  416 

Tvanoff,  415 

Massowah,  408,  416 

Metchnikovi,  417 

Nordhafen,  418 

of  Pestana  and  Bettencourt,  416 

Romanus,  416 
Vibrion  septique,  388 


INDEX 


605 


Virulence,  attenuation  of,  459 
exaltation  of,  461 
of  bacteria,  150 

Water,  bacteria  in,  135 

contamination  of  by  sewage,  138 
examination  of,  135 
supplies,  typhoid  bacilli  in,  345 
Weichselbaum  on  pneumonia,  198 
Weigert's  method  of  dehydration,  93 
modification  of  Gram's  method,  100 
Wertheim's  medium,  220,  226 
Whooping  cough,  bacteria  in,  423 
Widal  on  serum  diagnosis,  485 
Widal's  reaction,  synonym  for  agglu- 
tination of  b.  typhosus,  q.v., 
109,  337 
Winogradski,  23 
Winter-spring  fevers,  529 
Wolff  and  Israel's  streptothrix,  295 
Wooclhead  on  tuberculosis,  257 
Woody  tongue,  292 
Woolsorter's  disease,  311 
Wright's,  A.  E.,   calibrated  pipette, 

108 

diluting  pipette,  66 
method      of      counting      dead 
bacteria,  67 


Wright's,  A.  E.,   opsonic  technique, 

112 
vaccination  against  tiiberculosis, 

261 
vaccination    treatment   of   pyo- 

genic  infections,  194 
Wright,  J.  H.,  on  anaerobic  strepto- 

thrices,  293 
Romanowsky  stain,  107 

Xerosis  bacillus,  367 
Xylol,  93 

Yaws,  spirochsetes  in,  234 
Yellow  fever,  451 

bacteria  in,  452 

etiology  of,  452 

mosquitoes  in  relation  to,  453 
Yersin   (v.    also   Roux),    on   plague, 

435  et  seq. 
Yersin's  anti-plague  serum,  436 


Ziemanni,  spirillum,  550 
Ziehl-Neelseu  stain,  101 
Zoogloea,  3 
Zygote  (malaria),  328 


185888 


o <si -v 


